A rapid,inexpensive, and semi-quantitative method for determining pollen tube extension using fluorescence

Background

To identify plant genes involved in various key traits, QTL mapping is a powerful approach. This approach is based on the use of mapped molecular markers to identify genomic regions controlling quantitative traits followed by a fine mapping and eventually positional cloning of candidate genes. Mapping technologies using SNP markers are still rather expensive and not feasible in every laboratory. In contrast, microsatellite (also called SSR for Simple Sequence Repeat) markers are technologically less demanding and less costly for any laboratory interested in genetic mapping.

Results

In this study, we present the development and the characterization of a panel of 96 highly polymorphic SSR markers along the Arabidopsis thaliana genome allowing QTL mapping among accessions of the Versailles 24 core collection that covers a high percentage of the A. thaliana genetic diversity. These markers can be used for any QTL mapping analysis involving any of these accessions. We optimized the use of these markers in order to reveal polymorphism using standard PCR conditions and agarose gel electrophoresis. In addition, we showed that the use of only three of these markers allows differentiating all 24 accessions which makes this set of markers a powerful tool to control accession identity or any cross between any of these accessions.

Conclusion

The set of SSR markers developed in this study provides a simple and efficient tool for any laboratory focusing on QTL mapping in A. thaliana and a simple means to control seed stock or crosses between accessions.     

Using a multifaceted approach to reveal avian community responses to natural and anthropogenic effects in a fragmented Southern Mistbelt Forest system,South Africa

Landscape Ecology - Forest loss and fragmentation are major drivers of biodiversity decline globally. However, with the widely recognised notion that biodiversity is multifaceted, few studies have...     

The Nature Smart Cities business model: A rapid decision-support and scenario analysis tool to reveal the multi-benefits of green infrastructure investments

Incorporating natural spaces within urban areas has been shown to have multiple benefits. However, despite greening and adaptation strategies at different levels of government, progress remains slow with a lack of easy to use and comprehensive tools identified as key to overcoming this. This paper presents a co-designed tool with academic and local authority partners to demonstrate the ecosystem service benefits of small-scale urban green infrastructure projects. Through the tool, users can readily assess the impact of green infrastructure investments on the delivery of a selection of ecosystem services in the early stages of a project. Furthermore, the tool provides a standardised assessment of cultural ecosystem services’ contributions, as well as offering a method to score spatial designs on the impact on habitat for biodiversity. Use of the tool is demonstrated using a pilot study in Kapelle, the Netherlands. The results set out an overview of the impacts of the spatial design on estimated ecosystem service delivery. They also show the tool’s potential to add value in early project stages and as a planning and design tool, helping to maximise the benefits that can be achieved through green infrastructure design. Complementing these arguments with ball-park estimations on green infrastructure costs, the Nature Smart Cities Business Model aims to offer public sector officers the means to create a business case for green infrastructure measures, facilitating the translation from strategies to actual plans, thus benefitting green infrastructure implementation in the public realm.     

A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation

Background

Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life.

Results

Here we adapted Pro-Q^® Diamond (Pro-Q^® Diamond Phosphoprotein Gel Stain), a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT) method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q^® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1) on a thylakoid ascorbate peroxidase (tAPX), an established phosphorylation target in our earlier study.

Conclusion

The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays.

     

A simple method for rapid germination of pineapple seeds

Good germination in seeds of pineapple (Ananas comosus L. Merr) was obtained by exposing them to intermittent mist. Establishment of seedlings raised under mist was much better than after conventional practice. This method assures faster germination and obviates the need for the conventional practice of scarification and also the need for a carefully controlled environment.     

A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid

Background  

Salicylic acid (SA) is an important signalling molecule in plant defenses against biotrophic pathogens. It is also involved in several other processes such as heat production, flowering, and germination. SA exists in the plant as free SA and as an inert glucose conjugate (salicylic acid 2-O-β-D-glucoside or SAG). Recently, Huang et al. developed a bacterial biosensor that responds to free SA but not SAG, designated as Acinetobacter sp. ADPWH_lux. In this paper we describe an improved methodology for Acinetobacter sp. ADPWH_lux-based free SA quantification, enabling high-throughput analysis, and present an approach for the quantification of SAG from crude plant extracts.     

A simple,rapid method to isolate salt glands for three-dimensional visualization,fluorescence imaging and cytological studies

Background  

Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.     

A rapid method of fruit cell isolation for cell size and shape measurements

Background  

Cell size is a structural component of fleshy fruit, contributing to important traits such as fruit size and texture. There are currently a number of methods for measuring cell size; most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes or chemicals to release single cells. Neither of these approaches is ideal for assaying large fruit numbers as both require a considerable time to prepare the tissue, with current methods of cell wall digestions taking 24 to 48 hours. Additionally, sectioning can lead to a measurement of a plane that does not represent the widest point of the cell.     

植物非试管快繁技术在果树上的运用

该文综合阐述了我国当前果苗生产存在的问题与限制因素，分析了国外种苗生产的趋势，提出了利用新型的育苗技术——植物非试管快繁技术，实现种苗生产工厂化、自动化、产业化的生产模式。结合生产、科研实践，概括性地论述了快繁技术在果树种苗培育上的应用与优势，并指出了各种果树的快繁技术要点，展示了该技术在果树种苗快繁上运用的广阔前景与市场空间。     

梨叶片中9种多酚类物质的UPLC测定方法

【目的】建立超高效液相色谱(UPLC)—光电二极管阵列(PDA)技术检测梨叶片多酚类物质的方法。【方法】以成熟梨叶片为材料,优化色谱分离条件(流动相、检测波长和浓度梯度),并进行方法验证。【结果】流动相:0.5%甲酸(A)和乙腈(B);浓度梯度洗脱条件:0~1 min,0~5%B;1~6 min,5%~17%B;6~10 min,17%~25%B;10~11 min,25%~100%B;11~14 min,100%B;14~15 min,100%~0 B;15~20 min,0 B;检测波长:280 nm、325 nm和350 nm。利用该方法同时将9种多酚标准物质分离。多酚类物质的线性回归方程相关系数均在0.999 4以上,回收率均在83.39%~94.31%,精密度的相对标准偏差均小于4.77%。【结论】该方法准确、精密度高,适合梨叶片中9种多酚类物质的检测。     

甜瓜果实酸性转化酶基因反义表达载体的构建(英文)

应用RNA反义技术抑制甜瓜果实发育过程中酸性转化酶活性,促进蔗糖积累,从而为培育优质品种提供了可行的新方法。将已克隆到pMD18-T载体上的甜瓜酸性转化酶基因用BamHⅠ和HincⅡ双酶切,得到该基因编码区1038bp的cDNA片段,将其定向插入到植物表达载体pROK2的BamHⅠ/SmaⅠ克隆位点,构建了甜瓜酸性转化酶cDNA反义表达载体(Anti-MAI1)。采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。利用叶盘法转化烟草,经PCR和PCR-Southern杂交检测,证明此基因已整合入烟草的核基因组中。     

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

Background  

The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.     

Development of the natural environment scoring tool (NEST)

Natural environments (green and blue space) are associated with a range of health benefits, but their use is likely to be influenced by the presence of features, facilities and amenities and the condition/maintenance, or the natural environment quality. Most ‘quality’ assessment tools have focused on green spaces and their support for physical activity. This limits their utility for assessment of other natural environment typologies and uses (e.g., social, relaxation). We aimed to develop a tool for feasible, in situ assessment of diverse natural environments that might support a variety of uses, and to explore associations between natural environment quality and objectively measured amount of natural environment and neighbourhood-level socio-economic status (SES).This work was conducted as part of the PHENOTYPE project. Data were collected in 124 neighbourhoods in four European cities (Barcelona, Doetinchem, Kaunas, Stoke-on-Trent). The Natural Environment Scoring Tool (NEST) was developed using existing tools, expert input and field-testing. The final tool comprised 47-items across eight domains: Accessibility, Recreation facilities, Amenities, Aesthetics − natural, Aesthetics – non-natural, Significant natural features, Incivilities and Usability; typology-specific Overall Scores were derived.In total, 174 natural environments, covering a range of typologies, were audited. Mean time to complete NEST was 16 ± 28 min. There was good inter-rater agreement. Mean domain scores showed some expected patterns by typology (e.g., higher Recreation Facilities scores in urban parks and formal recreation areas; lower Amenities scores in natural/semi-natural areas). Highest mean Overall Scores were observed for areas of blue space and woodland, the types of area that often lack the recreational facilities or amenities that can be dominant in physical activity-focused audit tools. There was a trend towards lower natural environment quality in neighbourhoods of lower SES, with some inter-city variation. Correlations between NEST scores and amount of natural environment indicated higher natural environment in areas with worse access. We recommend further testing of NEST in other locations in relation to use and health outcomes.     

A rapid field method for measuring net assimilation rate-stomatal conductance relationship: a feasibility test using grapevine leaves

Based on the assumption that the response time of the photosynthetic apparatus to changes in light intensity is faster than the response time of stomata, a rapid field method for measuring the assimilation rate (A) stomatal conductance (g_s) relationship in wine-grapes is proposed. Leaves from the outer canopy of Vitis vinifera L. cultivar ‘Cabernet sauvignon’ were placed between two pieces of foam for 90 min to reduce light intensity and induce stomatal closure, while allowing water vapor diffusion to occur. Heat load was reduced by shading the foam with a piece of light colored, heavy paper attached to the foam with a 10 mm air space between them. When the cover was removed, A and g_s were measured several times until no significant changes in g_s were observed. The A/g_s relationships of the leaves were similar in spite of differences in the response time between leaves. Very few exceptions to the A/g_s curves existed, and these leaves were easily identified. This procedure permits an analysis of the A/g_s relationship without the complications of different plant water potentials that are normally used to achieve different levels of g_s.     

Development of a simple thermal time method for describing the onset of morpho-anatomical features related to sweetpotato storage root formation

A thermal time-based approach using growing degree days was used to describe the onset of morpho-anatomical features associated with storage root developmental stages in the sweetpotato cultivar ‘Beauregard.’ Initially, we calculated accumulated growing degree days corresponding to adventitious root initiation, the onset of storage root formation (appearance of anomalous cambium), and appearance of visible storage roots with localized thickening and pigmentation in sweetpotatoes grown under greenhouse conditions. Subsequently, we performed field calibration of this approach to verify the timing of these storage root developmental stages under natural growing conditions. Under field conditions, transplants required relatively fewer calendar days to achieve the thermal time corresponding to each developmental stage in comparison to greenhouse-grown plants. Further calibration of this approach for ‘Beauregard’ and other sweetpotato varieties grown in diverse environments will help enhance our understanding of the effects of management and agroclimatic variables on sweetpotato yield determination. This study demonstrates the advantage of using thermal time vs. calendar days in estimating storage root development in sweetpotatoes.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin

AIM:To investigate whether the bcl-2 antisense oligonucleotide increases the sensitivity of HL60 and K562 cell lines to daunorubicin.METHODS:IC50 for HL60 and K562 was determined with MTT method, the expression levels of Bcl-2 protein were assayed by immunofluorescence using fluoresce isothiocyanate labeling. In addition, apoptosis was detected by morphological observation and flow cytometric analysis of DNA fragmentation.RESULTS:It was found that the two oligonucleotides directed against the coding region and the translation initiation of bcl-2 mRNA, combined respectively with daunorubicin, inhibited expression of bcl-2 protein, increased apoptosis in HL60 and K562 cells, and decreased IC50 of daunorubicin significantly (P＜0.05). Compared to the antisense oligonucleotide directed against the translation initiation of bcl-2 mRNA, the antisense oligonucleotide directed against the coding region showed stronger effects in the aspects of increasing the sensitivity of HL60 cells to daunorubicin (P＜0.05).CONCLUSIONS:These two antisense sequences in the translation initiation and the coding region of bcl-2 mRNA increased the sensitivity of HL60 and K562 cell lines to daunorubicin in a sequence-specific manner.     

Enhancement effect of adenovirus mediated antisense c-myc gene and caffeine on chemotherapy of osteosarcoma cells to cisplatin

AIM: The cancer biology has showed that overexpression of oncogenes is responsible for the progression of human malignancies,antisense technology can block a certain gene expression.Caffeine has enhancement effect on chemotherapy of osteosarcoma cells to cisplatin,we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 base pairs in a reverse direction into adenovirus vector.Ad-Asc-myc and caffeine was used respectively or together to co-operate with cisplatin to treat the osteosarcoma MG-63 cells in vitro,and Western blotting,MTT,flow cytometry (FCM),electron microscope were used to evaluate expression of c-Myc protein,tumor cell proliferation in vitro,apoptosis and cell cycle analysis.RESULTS: Ad-Asc-myc was obtained with the titer of 2×10¹² pfu/L.Ad-Asc-myc down-regulated the expression of c-Myc protein,Ad-Asc-myc or caffeine enhanced the effects of 2.0,5.0 mg/L cisplatin on MG-63 cells.Moreover,Ad-Asc-myc combined with caffeine significantly enhanced this effects,not only on cisplatin-induced apoptosis,but also on tumor cells proliferation in vitro.The expression of bcl-2 was downregulated,bax were upregulated,while there was no change in the expression of E2F-1.FCM analysis showed that cisplatin treatment induced a block in S phase,and caffeine reversed this block and speeded up the progression of cells out of the S phase.Ad-Asc-myc induced obvious G₂/M phase arrest in transfected cells.CONCLUSION: Ad-Asc-myc combined with caffeine may enhance apoptosis-induced and chemotherapy effects of osteosarcoma MG-63 cells to cisplatin.     

越橘基因组DNA的快速提取及分析

为了从富含多酚、多糖及色素的越橘叶片中提取适用于分子生物学研究的高质量基因组DNA。以越橘幼叶为实验材料,比较了CTAB、SDS、高盐低pH值3种提取方法,获得了一种以CTAB法为基础的分离高质量完整DNA的简便、快速方法。用紫外分光光度计、琼脂糖凝胶电泳、RAPD-PCR、酶切等方法对获得的DNA进行了分析,结果表明,快速CTAB法所提取的DNA产量高、质量好,完全能够满足RAPD、PCR等分子生物学实验的要求。     

Three kinds of antisense oligodeoxynucleotide enhance the sensitivity of leukemia cell K562 to cisplatin

AIM: To investigate whether antisense oligodeoxynucleotides of hTERT、bcl-2 and c-myc could enhance the sensitivity of leukemia cell K562 to cisplatin. METHODS: The inhibiting effects of cisplatin and cisplatin plus antisense oligodeoxynucleotide on K562 cells were determined by MTT. RESULTS: The inhibiting rate of 20 μmol/L cisplatin to K562 cell is 17.17%±1.36% and it becomes 25.41%±1.77%, 26.18%±1.43% and 28.29%±1.05%, respectively, as combinated with antisense oligodeoxynucleotide of hTERT, bcl-2 or c-myc. CONCLUSION: These results indicated that antisense oligodeoxynucleotides of hTERT, bcl-2 and c-myc enhanced efficacy of cisplatin in K562 leukemic cells.