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,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.     

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 rapid,non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence

Background  

Analysis of survival is commonly used as a means of comparing the performance of plant lines under drought. However, the assessment of plant water status during such studies typically involves detachment to estimate water shock, imprecise methods of estimation or invasive measurements such as osmotic adjustment that influence or annul further evaluation of a specimen's response to drought.     

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 and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis

Characterizing plant genetic resources and their response to the environment through accurate measurement of relevant traits is crucial to genetics and breeding. Spatial organization of the maize ear provides insights into the response of grain yield to environmental conditions. Current automated methods for phenotyping the maize ear do not capture these spatial features. We developed EARBOX, a low-cost, open-source system for automated phenotyping of maize ears. EARBOX integrates open-source technologies for both software and hardware that facilitate its deployment and improvement for specific research questions. The imaging platform consists of a customized box in which ears are repeatedly imaged as they rotate via motorized rollers. With deep learning based on convolutional neural networks, the image analysis algorithm uses a two-step procedure: ear-specific grain masks are first created and subsequently used to extract a range of trait data per ear, including ear shape and dimensions, the number of grains and their spatial organisation, and the distribution of grain dimensions along the ear. The reliability of each trait was validated against ground-truth data from manual measurements. Moreover, EARBOX derives novel traits, inaccessible through conventional methods, especially the distribution of grain dimensions along grain cohorts, relevant for ear morphogenesis, and the distribution of abortion frequency along the ear, relevant for plant response to stress, especially soil water deficit. The proposed system provides robust and accurate measurements of maize ear traits including spatial features. Future developments include grain type and colour categorisation. This method opens avenues for high-throughput genetic or functional studies in the context of plant adaptation to a changing environment.     

A simple and efficient method for isolating small RNAs from different plant species

Background  

Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.     

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.     

Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

ABSTRACT: BACKGROUND: Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to only few plant species and often requires substantial equipment and facilities. The high content of chloroplasts and chlorophyll can impede downstream applications of transformed cells from green plant tissue. RESULTS: We describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima) for which no particular growth facilities or expensive equipment are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in all commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to a low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Transgene expression is detectable within 90 min post-transformation and lasts over several days. CONCLUSIONS: The simplicity of isolation and transformation renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multicolour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.     

A simple and efficient method for the long-term preservation of plant cell suspension cultures

Background  

The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.     

一种简便有效的香蕉小分子RNA提取方法

【目的】提供一种简单、经济、高效的香蕉小分子RNA提取方法,以满足RT-PCR、Northern杂交等分子生物学研究的需要。【方法】以巴西蕉(Musa acuminata L.AAA group,‘Brazilian’)的叶片、雄花、果实和根系为材料,利用改良的CTAB法,结合使用PEG8000分级沉淀DNA和大分子RNA,从而获得小分子RNA。【结果】琼脂糖凝胶电泳显示小RNA带型清晰,无DNA和大分子RNA干扰,说明小RNA质量较好。获得的小RNA经紫外光谱分析其A260/A280的比值在1.872~2.020,产量可达35μg·g-1。以提取的各组织小分子RNA为模板,利用茎环RT-PCR方法在香蕉不同组织小RNA中均检测到mi RNA156a,其扩增片断大小约为70 bp,且测序结果与预测的香蕉mi R156a序列一致。【结论】本实验提供了一种简便高效的香蕉小分子RNA提取方法,可满足RT-PCR、Northern blotting及小RNA文库构建等后续分子生物学研究的需要,为研究人员在实际工作中提供了更多的选择。     

A rapid method for quantifying landscape-scale vegetation disturbances by surface coal mining in arid and semiarid regions

Context

Quantifying landscape-scale vegetation disturbances by surface coal mining (SCM) is crucial for assessing and mitigating its negative impacts on the environment. Methods for detecting such disturbances in woody ecosystems exist, but these methods do not work well for deserts and grasslands in arid and semiarid regions because of their sensitive responses to precipitation variations.

Objectives

The objective of this study was to develop a new index to reliably detect the locations and spatial extents of SCM-induced vegetation disturbances in dryland regions in the face of fluctuating precipitation.

Methods

We have developed a vegetation disturbance index (VDI) that combines MODIS EVI data with precipitation data to detect vegetation disturbances by SCM on the Mongolian Plateau during 2000–2015. The VDI is computed by comparing vegetation production per unit precipitation for a given year with a multi-year mean, and by considering distances from coal-mining areas.

Results

Our results show that the VDI was able to adequately distinguish vegetation disturbances by SCM from climate-driven vegetation changes in five selected sites across the Mongolian Plateau.

Conclusions

The VDI provides an effective tool for quantifying the locations, spatial extents, and severity of vegetation disturbances by SCM in arid and semiarid regions.

     

一种适合于富含多糖和酚类物质的香蕉果实RNA提取方法

酚类化合物和多糖是影响香蕉组织RNA提取的几个主要干扰因素。研究通过在裂解液中加入聚乙烯吡咯烷酮去除多酚类化合物和利用水饱和乙醚去除多糖,改进了香蕉果实RNA的提取方法。该方法具有快速、简单等特点。     

A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress

We describe an efficient method for the rapid quantitative determination of the abundance of three acidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits the sensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantify the phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue.     

A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

Background  

The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs.     

A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

Background  

Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices.     

Protocol for rapid propagation,and to overcome delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L.

Single medium based efficient protocol for rapid propagation, and to overcome the delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L. through in vitro rhizome induction was achieved. MS medium with combination of 8.87 μM N⁶-benzyladenine (BA), and 2.46 μM indole-3-butyric acid (IBA) induced a mean of 6.2 shoots per explant. Addition of 11.7 μM silver nitrate to 8.87 μM BA and 2.46 μM IBA supplemented medium facilitated the highest number of shoots (mean of 8.3 shoots) as well as roots within 60 days. Subculture of isolated shoots on medium with the same concentration of BA, IBA and silver nitrate increased the number to a mean of 12.1 shoots. Silver nitrate enriched medium developed rhizome at the base of shoots. Increase of sucrose concentration (6–8%) in medium with BA, IBA and silver nitrate favoured the best rhizome development. Ninety five per cent of the plantlets survived in field conditions. The plantlets established in field without in vitro developed rhizome (from medium with BA and IBA) did not form rhizome even at 7 months after transplantation. Instead, they developed tuberous roots only. The plantlets with in vitro developed rhizome (on medium having BA, IBA, silver nitrate, and 6–8% sucrose), and that established from conventional way (through splitting of old rhizome) showed no difference in growth of the rhizome. The present study emphasizes the efficacy of silver nitrate and sucrose to develop rhizome in vitro, which enabled to overcome the delayed development of rhizome, and reduced yield of plantlets established in field without in vitro developed rhizome.     

A novel,repeatable, and non-destructive method to collect root xylem exudate from mature apple trees in an orchard and to compare its constituents with shoot exudate

SUMMARY

A method is described in detail to allow repeatable and non-destructive extraction of xylem exudate from the trunk of mature apple trees (Malus domestica Borkh.) by the use of reduced air pressure. The method allows extraction of sufficient uncontaminated exudate to determine and quantify constituents such as plant hormones, sugars, amino acids, and minerals. Analytical measurements from the first experimental year were conducted to determine the minimum volume of exudate required to obtain reliable values for various exudate constituents. Some constituents of root exudates differed compared to shoot exudates obtained by traditional destructive methods. Thus, shoot exudates may be less suitable if the influence of roots on the aerial parts of a tree is being studied (e.g., in relation to environmental conditions or horticultural practices such as root pruning, girdling, irrigation or fertilisation). The extraction method worked well between Spring and mid-Summer over 3 years. However, the procedure failed from the end of June, onwards. Possible reasons for this failure are discussed, and experiments which may improve lateseason sap recovery are suggested. Small modifications to the method should allow simultaneous, non-destructive extraction of root and shoot-exudates, permitting a more coherent picture of long-distance signals from root-to-shoot, and vice versa, and their co-ordinating significance.     

Expression of genes for two phosphofructokinases,tonoplast ATPase subunit A,and pyrophosphatase of tea roots in response to phosphorus-deficiency

Summary

Limited data are available on the levels of expression of the genes encoding ATP-dependent phosphofructokinase (ATP-PFK), pyrophosphate-dependent phosphofructokinase (PPi-PFK), tonoplast adenosinetriphosphatase (V-ATPase) subunit A, and tonoplast pyrophosphatase (V-PPiase) in response to phosphorus (P)-deficiency. Tea (Camellia sinensis) is a useful species in which to investigate adaptations to P-deficiency in higher plants due to its high tolerance to P-deficiency. Partial cDNAs encoding these four enzymes were isolated from tea roots. Thereafter, semi-quantitative RT-PCR was performed to analyse the levels of expression of these four genes in tea roots in response to P-deficiency. The results showed that P-deficiency decreased the levels of expression of the genes for ATP-PFK and V-ATPase subunit A, whereas not only did it not decrease, but it slightly increased the levels of expression of the genes for PPi-PFK and V-PPiase. This may contribute to the high tolerance of tea plants to P-deficiency.