Testing heat tolerance in the field

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Published on: March 21, 2013

Global climate change and localised human impact, such as waste disposal or fertilizer use, has and will continue to have an effect on the world’s flora, both natural and agricultural. Predicting this effect can be difficult, but it is important. If land managers and farmers know which species will cope well with change, they will be better able to make a decision about the species which will struggle under certain conditions.

If a species is well-researched, it may be possible to look for QTL associated with resistance to heat, drought, flooding, or other abiotic stresses, but of course this does not predict real-world responses reliably and in any case is not an option in all cases. In the lab or greenhouse under controlled conditions, a simple observation experiment can tell you the effects of various conditions on a plant, but again this is not an indication of in situ viability.

Buchner et al. published a method of determining the heat tolerance of plants in the field in this month’s Plant Methods (vol. 9:7). Heat was the only imposed variable in their protocol, so any environmental factors are included in the experiment. The group, from Othmar Buchner’s group at Innsbruck, made their own Heat Tolerance Testing System (HTTS) from a number of pieces of technical equipment, including the customized exposure chambers seen in the image above (Figure 5B in the paper). (more…)

Arabidopsis hydroponics video method

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Published on: March 8, 2013

One of the most frustrating things in lab-based research is trying to learn a new method from a paper. In my short time in the lab, I sometimes had to follow a trail of breadcrumbs back through several papers to find details of a single step in a protocol, on one occasion digging around in the library archives for a paper from an out-of-publication journal. Once the various reagents had been rounded up, I’d interpreted the protocol (What kind of ‘mixing’? How slowly is ‘slowly add’?), and had failed to accurately measure a solution that dissolved my pipette tips, all I usually had to show for my pains was a questionable precipitate and a lot of washing up – at least for the first attempt.

Researchers from three Australian research centres had similar problems with hydroponic systems described in the literature. Conn et al. designed their own hydroponic system for Arabidopsis and published it in Plant Methods (2013, 9:4) along with the YouTube video above, demonstrating exactly how the protocol works in practice. Plant Methods is a friendly journal for the intrepid researcher attempting protocols new to their research group. This paper is typical and has a comprehensive list of necessary reagents and equipment, and clear step-by-step guide with critical points highlighted.

If you need to grow Arabidopsis in a controlled environment to look at the physiology of the whole plant and you are unhappy with your current growth facilities, take a look at this paper on DIY hydroponics. It is quite a work intensive set-up (drill-bits are mentioned) but most of the equipment is cheap and easily come by.

Video Credit: Matthew Gilliham, via YouTube.

New Methods and Resources (II)

Categories: methods
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Published on: February 12, 2013

As promised, here’s part two of my selection of recently published plant methods and resources.

Nisar et al. (2012; Plant Methods 8:50) present a method for easy inflorescence stem grafting in Arabidopsis. I can’t vouch for its ease, but the typical clear Plant Methods format provides plenty of description and comprehensive materials and methods section as well as a step-by-step guide to their customised wedge-cleft grafting technique. The authors even provide a table of technical tricks for each step in the protocol.

Abraham and Elbaum (2012; New Phyt. 197:1012-9) present a method of quantifying microfibril angle in secondary cell walls. The method is technical enough only to be of interest to researchers who need to know the angle of secondary cell wall microfibrils – this is not a look-see ‘Friday afternoon experiment,’ as my old supervisor used to say. To get a full picture, scanning electron microscopy, small-angle X-ray scattering, raman microspectroscopy should all be used in addition to the new technique, which is based on customised polarized light microscopy and LC-PolScope, an imaging software.

Cui et al. (2013; Plant Phys. 161: 36-47) demonstrate that the Tnt1 retrotransposon is a powerful tool for functional genomes in soybean. 62% of insertions from Agrobacterium-mediated transformations using a Tnt1 vector were into annotated genes, indicating the Tnt1 element preferentially inserts into protein-coding regions.  Multiple insertions occurred per transformation, and the transposons did not jump under normal growth conditions. The authors obtained the Tnt1 transposon from plasmid pHLV4909, which contains the entire sequence, and cloned it into the binary vector pZY101 for the transformations.

New Methods and Resources (I)

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Published on: February 5, 2013

On this blog, I highlight a new method or resource pretty regularly. I used to work in what I think is a fairly normal UK plant science lab, so I try to comment on aspects I would have found useful to know about, for example if the method requires a machine not every lab has, or if it is unclear about anything. However, there are many, probably excellent, new open software and techniques which I don’t highlight on the blog because I am completely unfamiliar with their background.

For today, here’s the first part of a round-up of plant methods and resources published over the last few months. If you have used them, feel free to let me know how they worked in the comments, or through email or Twitter. And if you would like to review a method or resource for this blog, please get in touch!

iRootHair is a free, online, curated, expandable database of root hair genomics. Kwasniewski et al. (2013; Plant Phys. 161:28-35) built the database, which currently includes information about 153 root-hair related genes. The majority of the genes are from Arabidopsis, but maize, rice, tomato, and barley genes are also included. There is a page showing figures of various root phenotypes, which users can click through to see the genes associated with a specific phenotype; and a similar one for root processes like tip growth. (more…)

Analysing phenotypes and measuring callose

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Published on: January 9, 2013
Part of Figure 7 from Green et al., showing the an example of Phenophyte output.

At the end of last year, you may have missed two useful publications from Plant Methods which use new free online tools to make your life easier.

Phenophyte can help you measure 2D areas quickly and accurately. It was described in November’s Plant Methods by Green et al., a team mainly from  Columbia, USA. Users chose if they want to analyse indivudial images, compare before/after images (as shown in the figure to the left), or analyse a timecourse. They then upload the images – the upload tool allows up to 2GB or 500 images, and sequential uploads are possible if required. The computational results can be previewed before submitting the job. When processing is complete, the user will be emailed a link to the results, which must be downloaded within a week. The manual provides detailed tips on how to take the photographs to upload, and the guidance is standard with the exception of the use of a colour/size checker (for example, this one), and the interface is straightforward and friendly.

Figure 5 from Zhou et al., showing the CalloseMeasurer interface and output.

A more specialized application is CalloseMeasurer, from the Robatzek group at The Sainsbury Laboratory. Zhou et al. describe a piece of software for quantifying callose deposition with enough accuracy to quantify the growth of filamentous pathogens within a plant by recognising the spreading network of callose deposition caused by the pathogen. The paper is heavy on technical detail, but guides readers through using CalloseMeasurer in the ‘Image Processing’ section of the paper. Users must have Acapella software installed, and they simply drag and drop the CalloseMeasurer script into the application window and start using the programme.

Phytotracker

Categories: methods, resource
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Published on: November 6, 2012
Fig. 4 from Nieuwland et al. (2012), showing the Phytotracker labels

If you have ever been frustrated by a less than clearly labeled seed stock, not knowing what the green and yellow dots mean, how long its been in that drawer, or which generation it is, GARNet Chair Jim Murray’s lab in Cardiff have worked out a system that will help.

Phytotracker is a system that organizes your seeds for you. Of course it depends on people recording the tray number and parent lines in the database, and correctly labeling the seed stocks and plasmids in the lab. However, once you’ve done that, you can forget about it because Phytotracker does the remembering for you – everything from which plasmid was used for the transformation to when to harvest the seeds.

The system is well explained in the paper, which was published in Plant Methods in October. If you want to try the system out, you’ll need Filemaker Pro (version 8 or later), or for a fully networked solution Filemaker Pro Advanced (version 8 or later: currently Filemaker Pro is version 12). Your University may already have a site licence! You’ll also need printers in your growth rooms and labs to print labels for the trays, plants, and seed stocks. Commitment from everyone in your group is essential – this system would fall apart if you have a regenade group member who insists on labeling with autoclave tape and a Sharpie. It has been successfully used in Cardiff for five years though, so it looks like a system that is worth committing to.

Highlighted article: Jeroen Nieuwland, Emily Sornay, Angela Marchbank, Barend HJ de Graaf, James AH Murray (2012) Phytotracker, an information management system for easy recording and tracking of plants, seeds and plasmids. Plant Methods 8:43

Download Phytotracker here: http://sourceforge.net/projects/phytotracker/

 

Method: New and improved multiplex PCR

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Published on: August 30, 2012

Highlighted article: Daxing Wen and Chuqing Zhang (2012) Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments. Plant Methods 8:32 (Online preview) doi: 10.1186/1746-4811-8-32

Background

Multiplex PCR allows amplification of multiple targets in a single PCR experiment. It is possible to amplify several sections of a single template, or to amplify different templates using a number of primer sets. If there are multiple primers in a reaction, it can be difficult optimise the PCR reaction to maximise the efficiency of every primer, and it is likely that some cross-hybridisation and mis-priming will occur.

Figure 3B from Wen and Zhange (2012). A comparison of multiplex PCR (Lanes 1-4) and universal multiplex PCR (lanes 5-8), using the same primers with universal adaptors. The band intensity from traditional PCR is very variable, but it is consistently strong when the universal adaptors are used. 

Image credit: BioMed Central

 

The Method

Wen and Zhang from Shandong Agricultural University have devised a way around the inconveniences of multiplex PCR to develop a universal multiplex PCR method. ‘Universal adaptors’ are linked to specific primers, making the annealing temperature of the adaptor-primer structures 70°C. (more…)

Identifying mutations in Arabidopsis – a faster, cheaper method


Highlighted paper: Liu, McCormack and Sheen (2012) Targeted parallel sequencing of large genonmic regions for identifying mutations in Arabidopsis. Plant Methods 8:12

Kun-hsiang Liu, Matthew McCormack and Jen Sheen from Harvard have developed a PCR-based method of identifying mutations in Arabidopsis. It is cheaper and easier than traditional methods of identifying mutations, using bench-top PCR and a new user-friendly method of bioinformatics analysis using web-based resource Galaxy. Liu et al. estimate that using their method to identify a mutation mapped to a 550kb genomic region will cost roughly US$500, a fraction of the usual ten thousand dollar cost of currently used methods of mutant identification.

Liu et al. tested the new method of identifying mutations by searching for new nitrogen response genes. They generated an Arabidopsis thaliana line in which LUCIFERASE was driven by the promoter for nitrogen response marker NIR. Using EMS-mutagenesis, the team made 25 000 mutant NIR:LUC lines and identified seedlings that were nitrate insensitive (nis) or showed nitrate constitutive response (ncr).

When the lines were made, the phenotypes were identified and a second generation was grown. Three second generation lines – ncr1, nis1 and nis2 – were selected for further investigation.

Liu et al. used their novel TPSeq method to locate the mutations causing the ncr1, nis1 and nis2 phenotypes. (more…)

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