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Questions related to Plant Molecular Biology
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I performed micrografting on mutant for ABA transport and most of them didn't succed. I'm considering that the mutation might have hindered the vascular regeneration. Do you know how ABA could affect vascular regeneration?
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Nicolò Maria Villa ABA regulates root growth, especially defining the directions in which the root system spreads. Because the development of vascular elements is important for root growth, ABA necessarily also affects the development of vasculature. There might be a specific interaction between the particular ABA transporter in your research and some element of the vascular development cascade - that interaction may or may not be already researched, so you would need to take a thorough look at the literature. Maybe you might start from the following:
And, possibly:
and see where that will lead you.
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I have used RefFinder (www.heartcure.com.au/reffinder/) to rank and help me select various reference genes for my qPCR experiments. The reference on their website (Xie et al, Plant Molecular Biology) does not truly go into their algorithm so I am looking for a better reference to cite when I publish. Does anyone know of one?
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Hello Ellen Ingolfsland re: "citation for the RefFinder reference gene stability algorithm?" (I am not a specialist in your field) The following may duplicate what you have or it may be of use:
re: Refinder from SciCrunch Registry:
FYR: See:
  • Spiegelaere, W. D., Dern-Wieloch, J., Weigel, R., Schumacher, V., Schorle, H., Nettersheim, D., Bergmann, M., Brehm, R., Kliesch, S., Vandekerckhove, L., & Fink, C. (2015). Reference Gene Validation for RT-qPCR, a Note on Different Available Software Packages. PLOS ONE, 10(3), e0122515. https://doi.org/10.1371/journal.pone.0122515
  • “To facilitate the use of multiple reference genes, a series of algorithms were developed to enable a comparison of the stability of the reference genes. Three of these algorithms, i.e. BestKeeper, geNorm, and NormFinder, have been incorporated in free to use Excel based software packages [4,7,9]. The development of these packages, along with newly developed R-based packages using these algorithms has resulted in a rise of research papers in which reference genes are compared. Recently, these algorithms were combined in a free to use web-tool (RefFinder) that, in combination with a fourth comparison termed the comparative CT method [10],”
Cheers,
Leo
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In plants, phyohormones induced responses generally see the binding of transcription factors to the special motifs present in the promoter of a downstream gene. After a few preliminary results, I was checking if the well known DNA binding element is present in my gene's promoter or not. Although I found a few variant of the original motif sequence in the promoter region, the exact motif which I was looking for I found is present in some +100 base pairs down to the start codon. In my understanding as far as I know, the DNA binding elements (motifs) are generally found in the upstream of the protein encoding region (promoter usually). But does anyone knows about cases where transcription factor binding sites, the DNA motifs are also present inside the gene itself?
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There are many, many examples of TF binding sites in the gene body. After the first whole genome ChIP experiments (ChIP-chip and ChIP-Seq) , it became obvious.
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Hello everyone!
My protein which has a GFP tag is well expressed in nuclei. To visualize cell boundaries in different layers of the root sample, I am planning to counter stain my samples with propidium iodide and DAPI.
I will be taking images using 3i spinning disc confocal microscope.
I want to know if anyone has a working protocol for such settings.
Earlier, I tried Hoechst 33342 to stain nuclei but it appears that even incubating for 30 minutes, the stain was not able go inside the cells as the cell boundaries were clearly visible during microscopy. I tried DAPI as well, but the background was very high even after 3-4 washes and the intensity of nuclei was not great.
I will appreciate if you could share with me your experiences and suggestions regarding my question.
Thanks!
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Hi, are you planning to use the propidium iodide for both cell boundaries and nuclei?
If your GFP tag is well expressed in the nuclei, you do not need to stain it again. But if the fluorescence signal is not as bright as you want it to be, open the pinhole for more light to reach your sample.
I have used propidium to stain the cell boundary of Arabidopsis roots in the past, and it worked brilliantly. I stained the root for just 5 minutes using the recommended concentration on the bottle.
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So I have always used Hygromycin/Kanamycin selection for Arabidopsis but this is my first time I am using methotrexate for selection of T0 transformed plants. Plants with hygromycin resistance I generally keep in dark and in3-4 days the one with resistance grows well.
I found only 2-3 papers where methotrexate has been used and it was not clearly mentioned that if there are more conditions for methotrexate selection other than just ading methotrexate to MS medium.
The working concentration I am using is 0.1mg/L.
Please let me know if you have experience with Methotrexate selection.
Thanks in advance.
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Hi there, I've used MTX regularly and have created several mutagenic Arabidopsis lines using this selection. It's very effective and easy to use. Just place it in the medium at a final concentration of 100 ng / mL* and sterilise / plate your seeds from transformed plants as you normally would.
MTX affects root growth by inhibiting the function of Dihydrofolate reductase, part of folate metabolism pathway, and the resistance gene is a variant form of the DHFR gene that is resistant. So sensitive seedlings will germinate and will start to grow a shoot and root, but will stop when the root is ~1 mm long. Resistant individuals will produce a healthy full-length root - quite easy to find in the plate.
* this is 0.1 mg / L as you say: to get to this low concentration, I dissolve the MTX powder in DMSO to a final concentration of 10 mg / mL (should be strongly yellow), then make a working stock by a 1:100 dilution to 100 ug / mL (weakly yellow). Then use this working stock at a 1:1000 dilution in the media (so 50 uL in 50 mL media) to final concentration of 100 ng / mL. Make the stocks fresh, it doesn't seem to work so well when it's been frozen / thawed. Good luck!
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CTAB Extraction Buffer (1.4 mmol m-3 NaCl, 20 mol m-3 ethylene diamine tetra-acetic acid (EDTA), 100 mol m-3 Tris-HCl (pH 8.0) and 0.2% (w/v) beta-mercaptoethanol)is not working. Any suggestions?
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They are many good silica-based column kits such as NucleoSpin Plant II (M-NAGEL), DNeasy Plant Mini Kit (QIAGEN)...
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Agrobacterium culture OD?
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Actually, the OD measurement record of Agrobacterium depends on the type of tissue or explant and method employed, but mostly OD600 from 0.2-0.6 can be used.
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Is BRASSINOSTEROID INSENSITIVE 1-Associated receptor Kinase 1 (BAK1) a plant resistance (R) gene?
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Brasinosteroids, defined as the sixth plant hormone after the classic plant hormones auxin, gibberellins, cytokinin, abscisic acid and ethylene, are analogous to animal steroid hormones in structure. BR-insensitive (BRI) is the receptor for BR. BRI1-associated kinase 1 (BAK1) is another enzyme.
BRI1 and BAK1 interacted with each other in vitro and in vivo, which contributed to BR signalling 
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As you know, Monstera is very popular house plant and some has unusual mutation with reduced number of chlorophyll and in some cases, chlorophyll pigments completely lost in leaves. Is it possible to make chlorophyll degradation in leaves under lab conditions?
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Can the hypersensitive response occur as part of PAMP-Triggered Immunity (PTI) as well as Effector-Triggered Immunity (ETI)?
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Dear @Ruby Metzner
The ubiquity of hypersensitive response among higher plants despite its costs suggests that it is an extremely effective component of the plant immune system. Please check the following links, and attached pdfs; hope, these could be useful to you.
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Are resistance (R) proteins (encoded by R genes) involved in pathogen Avr protein recognition/interaction only? Or do genes associated with plant immune signalling or other pathogen defence mechanisms also fall into the category of R gene? For example pathogeneis related (PR) protein encoding genes or genes involved in salicylic acid signalling?
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Most identified R genes are polymorphic in plant populations, which led to their initial characterization and use in plant breeding programs. However, individual plants have up to a few hundred R gene analogs that make no identified contribution to resistance. Many of these R gene analogs are also fixed in plant species and are thought to contribute to non-host resistance. After more than 25 years of R gene cloning, R genes have been classified by Nine Mechanisms, details of which may be accessed at:
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Are resistance (R) genes expressed in response to pathogen attack - or only defense genes - as part of the plant immune response? R proteins act as receptors to recognize pathogen effectors and an interaction between R protein and effector stimulates Effector Triggered Immunity (ETI) which itself involves the expression of defense related genes. However, are R genes also expressed as part of ETI?
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Dear Ruby Metzner Plants have developed a complex defense system against diverse pests and pathogens. Once pathogens overcome mechanical barriers to infection, plant receptors initiate signaling pathways driving the expression of defense response genes. I have attached some PDFs; hope these will provide useful insight regarding answer to your question.
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Hi everyone!
I have a few lines of a particular gene of which the earlier (T2 ratio) are not known. I want to do antibiotic selection of these seeds and I want to know which plants of next generation (T3) are going to be homozygous. Is there an easy way to confirm number of T-DNA insertion of my gene present in genome of individual plants? Like some PCR strategy.
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I used a 3 primer method for identifying the insertion of my gene in Brachypodium T-DNA mutants , the protocol was given on the T-DNA mutant website.
They must have a protocol for Arabidopsis on the TAIR or SALK website, did you check it?
Briefly it mentions using your Gene specific primer with the forward or reverse T-DNA primer. In one reaction you add both T-DNA forward+ reverse+ your Gene specific primer. I have attached the protocol for your reference, it worked perfectly after the PCR on the gel you will see one band for homozygous insertion and two bands for heterozygous insertion.
Hope it answers the question, feel free to ask any further questions.
Best of luck Avinash Sharma
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Is there a database of presently identified grapevine disease resistance loci?
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Dear Ruby Metzner I have attached very useful papers from world class journals; hope these will be useful, and serve your purpose.
Best wishes, AKC
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for oxalate, i used 1g sample, 75ml 0f 3M H2SO4 to soak it. filtered and took 25ml aliquot and warm and titrate against 0,05M KMnO4. (1ml KMnO4=2.2mg).
for phytate, i used 2g sample, soak with 100ml 0f 2%HCl, filter and took 25ml aliquot and to it, 53.5ml dH20 was added. 10ml 0.3% ammonium thiocyanate soln was added and titrated against standard iron III chloride containing 0.00195n iron/ml.
 how do i determine these values in mg/100g?
thanks. swift response is anticipated
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Are plant pathogenesis-related protein genes, mlo genes, and SNARE protein defense genes classified as resistance (R) genes? Or do they fall into a different category?
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Yes protien genes,mlo genes and SNAR protein classified resistence R gene
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How well understood is pathogen response MAPK signalling in plants?
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Just download both papers and check references therein
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Are pathogenesis-related proteins and antimicrobial peptides encoded by plant resistance genes (R genes)?
I'm unsure if the genes for these proteins/peptides are types of R genes?
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How does MAPK signalling work in plants in response to pathogen infection?
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This topic is well explored and there is a huge literature resource and review articles available. You just need to find and focus on reading and understanding it.
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Dear Researchers,
        I am doing transformation in cotton plant. Now I want to check GFP expression in transformed tissue. Can anyone suggest a standard protocol for preparing tissue sample to visualize GFP under fluorescence microscope? Thanks in advance.
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Hi all,
In my lab we are designing some acute osmotic and salt treatments in plants of a endemic tomato variety to analyze the relative transcript levels of different genes by qRT-PCR at different times. One of the discussion we are having is how to perform the sampling. In one hand, some believe that the best is to pool samples and then perform the RNA extraction (3 plant per pool and 2 pool) and in other hand some believe in perform the RNA extraction and qRT-PCR experiments in each individual without pooling samples.
What do you recommend is the best approach?
Thanks!
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I believe that qRT-PCR of the genes of each individual wouldn't give you many answers. As already stated it will bring a lot of variation in the experiment. It would be more sound to do some pooling and make sure you've got at least 3 biological replicates for each of your treatments. For example you could pool x individuals together into one pool and do that for at least 3 pools. I usually do qRT-PCR on individual plants if THAT plant has a special phenotype that others might not have but I'm very careful in interpreting these results.
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Hello everyone!
I am working on a gene from Arabidopsis. Initially using the GUS staining I found the reagents and the conditions which altered the expression of my gene differently in shoot and root parts.
To further check the expression of gene at mRNA level I used the same set of conditions and did RNA isolation, cDNA preparation followed by RT-PCR. (primer efficiency were checked and RNA quality was monitored).
Please look at the image I have attached.
You could see that the untreated (control) whole sapling shows to have at least 1.5 fold upregulated gene expression in compare to NaCl and ABA treated whole samplings. Whereas, the root of the control (untreated) sampling shows approx. 2 fold reduction in gene expression in compare to NaCl and ABA treated root samples. The shoot samples of control (untreated) and treated (NaCl and ABA) shows not much difference.
How it could it be possible that the control (untreated) whole saplings have higher gene expression in compare to treated whole saplings when the gene expression levels are higher in the root samples of treated plants in compare to control root samples?
in all treatments, intact saplings (root + shoot) were incubated in different reagents for a defined time length. The roots and shoots were separated after the incubation period was over and RNA isolation was performed.
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Hello Avinash
Maybe you could check whether the housekeeping gene you used for normalization is stably expressed in different types of genes. If not, then that might be the cause for your unexpected results, since it is not recommended to normalized with genes with unstable expression.
You could have look this article. It provides some suggestions for reference genes.
"Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis"
With regards,
Luke
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I want to add 1 ug or (1000ng ) of Plasmid DNA in a reaction mixture and add water for a final volume of 50 ul. my plasmid DNA a concentration of 68.47 ng/ul in which I have total DNA volume 25ul. How much ul should i have been used into my mixture? dear any one help me. Thanks in advance
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You should not add 35.4 ul of H2O. Because you have other components to add.
14.60 uL (plasmid) + other components + water = 50 uL
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Nanoparticles (NPs) are characterized by their small size ess than 100 nm. They are able to bind the DNA through electrostatic interactions .
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Does anyone who know how many genes control parthenoecapy? Previous study of satsuma mandarin as seed parent, the result in favors of at least two co-dominant gene govern parthenocarpy. The detail function for each gene related to parthenocarpy is not clear. How should we consider the mechanism of parthenocarpy in citrus?
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Its a good question!
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I woul need a good journal to publish next generation sequencing given snp data of a plant gene.
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You may submit polymorphism data to Polymorphism journal located at
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I'm looking for an alternative method from the Qiagen RNeasy Plant Mini kit to isolate total RNA from Cotton root tissue. 
I'm considering using the TRIZOL reagent to hopefully get a higher RNA yield, but I've read that TRIZOL extraction produces RNA with a high level of carbohydrates (and thus a low 260/230 ratio). This would produce a sample that is far too impure to perform cDNA synthesis, so it's important that whatever method I use either produces RNA isolates with a high 260/280 and 260/230 ratio after extraction, or yields a high enough RNA concentration that I can do multiple ethanol washes without loosing too much of my isolates.
Does anyone have any experience using the TRIZOL reagent to extract RNA from Cotton tissues, namely the root systems?
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Hi Walker Maffit , did you get a chance to make this work? I know your post has been a while. I am curious about what worked for you. I am researching RNA-extraction methods from cotton roots currently, would like to hear people's successful procedures.
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Can this be accomplished using Cas9 and two sgRNAs targeting genomic sites over 100 kb away from each other? I want to do this in a plant that is transformable, but for which there are no reports of using CRISPR. If it is feasible, what range of editing frequency can I expect to achieve? Any literature sources on the use of gene editing for long deletions in plants generally?
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Hi Chris,
You can find some information from this reference. They could produce 115-245 kb deletions.
"Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice" Zhou et al., 2014. Nucleic Acids Res. 2014;42(17):10903-14. doi: 10.1093/nar/gku806. Epub 2014 Sep 8.
Good luck!
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Dear Colleagues/Editors/Reviewers,
Comment those journals which are going to receive its first impact factor in JUNE 2020 and currently indexed in ESCI, SCOPUS, PubMed, etc.
Journals must be related to plant physiology, plant biology, plant sciences, agriculture, and biotechnology, etc.
Note. Please don't comment any predatory journals. Thanks in advance.
Regards
Ali
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hello,
I got data on 25 germplasm lines for flowering time. I want to plot box plots of best linear unbiased predictors with error bars (either by taking SE or SD) by taking flowering time on X axis and germplasm lines on Y-axis. The three replication data for the germplasm lines and check variety for flowering time was recorded and i want to show the variability present in the flowering time.
Kindly suggest the  best software to do this.
ashok
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Hello,
I have some small secreted proteins which have the ability to move from plant to fungi. As they can move between plant and fungi, I assume these small secreted proteins have some domains responsible for their movement such as export and import domains. I am planning to generate a series of deletion constructs to identify domains that are responsible for protein movement. What might be the good experimental strategy to identify the domains? Is there any good system or experimental strategy that I can use for quick testing of my deletion constructs?
Thanks in advance
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Indeed the kind of experiment you use is too specific for general domain predicting tools (unless you know that there is a particular protein domain already observed to play a role in this process).
Despite that, testing deletions in silico is not so simples, especially if you want to make several deletions. One way I can think of is only possible if you have your protein PDB available. You can use Cytoscape [https://cytoscape.org/], and the RIN Analyzer plug-in [https://rinalyzer.de/]. RIN Analyzer created an interaction network between residues, allowing you to observe your protein as a network of nodes and edges. You need to read about the plug-in because it has tons of features and works with different other tools.
Then, you can use Interference [https://apps.cytoscape.org/apps/interference]. Interference allows you to create virtual knockouts, which reflects on a given set of topological parameters (You might need to send an e-mail to get it from the developers).
Please, check the version of Cytoscape to use both plug-ins because Interference is old. I also highlight that the use of these tools is an old protocol I tested, which provided some good results in a Protein-Protein Network, but I didn't test it in a Residue-Residue network. I don't know how it holds nowadays or if the developers still provide access to Interference. Nevertheless, this is a method I know, which is fast after you understand the tools.
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Does anyone know a good protocol to isolate RNA from cucurbitaceous seeds? We are trying to detect viral RNA in seeds by using the silica RNA extraction method but we cannot see any RNA when the samples are electrophoresed in 1,5% agarose gel. Any ideas?
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Hi All,
I am looking for several plant expression vectors (pML-BART, pART27 and pART7). Could anyone please tell me where I can find these vectors? I have searched in google but did not get any source where I can buy them.
Thanks,
Dr. Mahmudul Hassan
Postdoctoral Research
Oak Ridge National Laboratory, USA
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Hi , this website might want to contact them , they make plasmids according to order.
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Recently I work on Isolation & Identification of rhizobia bacteria of chickpea & groundnut.I want to know which rhizobia bacteria can create nodule of chickpea & groundnut/peanut.Please anyone help me so I will gratitude to you.Advance thanks.
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Hello,
Can anyone please provide me a detail protocol for coating gRNA and Cas9 protein onto gold/tungsten particles for particle bombardment in plants.
Thanks!
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It is quite challenging. And I agree with Yuan-Yeu Yau that the details are always missing in the papers. As far as I see it in my work is that simply mixing of Gold with RNPs by pipetting does work. When coating it works better when u dry them at 4°C. But even if u do everything very carefully it works in one shot and with the other not. To be honest the particle gun itself is one of the most shitty machines I ever used. Does someone now found some method which finally worked?
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Dear all
i plan to extract the mitochondrial DNA and RNA from plant tissues to identify and analyse the process of RNA editing.
in this case i have been searching online but there seems to be more protocols with a numerous reagents and steps.
i was wondering if any company like Qiagen, Thermofisher or.... offer any kit which is fast and reliable so that i can buy.
regards
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I am looking for methods (or kit) for mitochondrial RNA isolation. I need to go for RNA-seq. Could you suggest me in this regard.
Thanks
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Hello Everyone!
I am new to plant molecular biology and i have a doubt. Is it always necessary to clone a plant gene in an entry vector (such as pJET, pGEM®-T Easy vector) before cloning into a destination expression vectors such as pCAMBIA? what are the advantages? Thanks in advance.
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1. I would not call plasmids such as pGEM-T vector 'entry vector'. It is just a regular vector. You stick your gene into those vector, so you can multiple them (plasmid with backbone and your gene) and get enough plasmid material for next experiment. pGEM-T Easy vector can also be used for sequecing your gene after you ligate your gene into it.
2. When you talked about 'entry' and 'destination' vectors, it lets people think about the Gateway system, which use such terms.
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I found a paper on the plant journal. In BiFC, the authors fuse YFPN to C-terminal of a plasma membrane protein called RSL1 (at the outside of the cell). And they fuse YFPC to a soluble protein (in the cytosol, ABA receptor in this paper). The results show that there are interaction on plasma membrane between the two protein. The author said: "The TM domain of RSL1 is only 20 AAs large and there are a few AAs behind. We guess the GFP protein does not cross completely the membrane to the outside face of the cell."  How can GFP not cross completely the membrane? What does this mean? Is this possible?
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Hello Jen, Good question, Can you share the doi OR link for the paper
I my case, for one of my interactors, I have a nuclear export signal
Hence I see interactions sometimes in cytosol
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i am doing molecular characterization of citrus germplasm, Presently ihave started using some of the SSR markers published in Citrus sinesis genome paper. Presently i evaluating the PCR products on 4% metaphor gels. i am getting bands of different sizes and in some cases present and absent type of bands. SInce i am amateur in scoring molecular markers. i want to know is there any article published explaining how to score a SSR marker for further analysis. thank you 
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While using NTSYS Pc software for divergence analysis, the SSR data scored on the basis of presence and absence as well as molecular size difference have to be arranged into binary matrix. So each allelic variant recognized on the basis of molecular size difference is considered as marker and scored for its presence and absence and then subjected to divergence analysis.
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Best branded machines for my research work for the following instruments
PCR
Gel Documentation system
Cooling centrifuge
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T. Leon Stephan Raj Now a days there are tens of different companies with 100s of good products which are competitive in performance and prices. And there is no difference if you are using those machines for plant or any other molecular work. So, it depends upon you to choose what company suits you in terms of after sales services and prices.
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Use of Biochar for plant infecting virus
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Interesting..
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Hi, I'm currently writing my paper, and I have to compare some DNA sequences. But I found some samples were already been analyzed,
so I used analyzed data from NCBI and my own new data to comparison.
Now I want to state clearly that I used their data in my paper,
what is the best way to do it?
writing their GeneBank accession No. and put the papers in my references?
I'm worried if that is not enough. Should I contact the authors of the paper?
Help me with some experience! Thank you !
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There is no specific consensus on format for citing NCBI information, but a useful guide can be found here: https://www.ncbi.nlm.nih.gov/geo/info/linking.html
That is for NCBI's GEO database but the recommendations are useful for most facets of NCBI. There are expandable sections on "Citing the GEO database," and on "Citing your GEO submission," and on "Citing data you find in GEO" For example, pasted below is the section on citing data in NCBI GEO:
In addition to citing one of the GEO database papers listed above for general use of GEO, if applicable, you should cite the original paper and the accession number used to identify the experiment(s) in GEO. That way the original generators of the data sets will get credit and allow readers to locate the source. The original paper (if available) is listed under the 'Citation' section of the Series and DataSet records.
Examples:
  • "Smith et al., 2006, recently performed microarray experiments investigating effect of compound X on neurons (data accessible at NCBI GEO database (Edgar et al., 2002), accession GSExxx)."
  • "In March 2008, a search of the GEO Profiles database (Barrett et al., 2006) revealed that Gene X is upregulated in response to compound Y (GEO accession GDSxxx; Smith et al., 2006)."
For another example, pasted below is the section on citing submissions:
"The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSExxx (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSExxx)."
We strongly recommend that submitters cite their Series accession number (GSExxx) because that record summarizes the experiment and includes links to all other relevant data.
Also, NCBI requests that everyone who cites anything from GENBANK also cite the current GENBANK reference itself. Look through that paper for more citation suggestions (most recent version is 2017).
Dennis A. Benson, Mark Cavanaugh, Karen Clark, Ilene Karsch-Mizrachi, David J. Lipman, James Ostell, Eric W. Sayers; GenBank, Nucleic Acids Research, Volume 45, Issue D1, 4 January 2017, Pages D37–D42, doi:10.1093/nar/gkw1070
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In VIGS we silence genes but i wish to know if the silencing signal spreads in root or not. Putting it in other way, are silencing signals by VIGS or dS RNA degradation spreads equally from top (shoot/leaf) to bottom (roots) and bottom to roots (consider a RNAi construct in hairy root or VIGS) in plant systems.
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Nature Communications Vol 9, Article Number: 3107 (2018) entitled "
Gating of miRNA movement at defined cell-cell interfaces governs their impact as positional signals" explains a lot of this question.
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I have used "Floral dip" method for Agrobacterium-mediated
transformation of Arabidopsis thaliana, and it has always worked well. But this time, after the Floral Dip, my plants all died on the next day, and I can't find the reason.
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Hi Rui,
You might have kept the plants under pressure for a long time or you might have given too much Agrobacterium dosage. Sucrose and Silwet conc looks fine though. Keep the plants in dark for 2 days wrapped in moist condition. It might recover.
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For example the large carpenter bees can visit Calotropis or other wild bees visit Peganum. I would like to understand how the bee deal with these plants and is the nectar of these plants contain the same toxic contents of the whole plant? 
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Thanks a lot for this great clarification, although I left Saudi Arabia 3 years ago and the questions was to figure out how larger carpenter bees is adapted to get nectar from Calotropis procera. The stems of the plans also were used by bees for nesting. I may try to find this work again when I will be back to Egypt after my research visit to Hungary.
Thanks again Christopher and I wish for you all the best.
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Rose, RNA, real time
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i am also having a lot of trouble in RNA extraction from Rose tissues (leaves, flower buds, etc). I have used pBiozol, but still i haven't been successive in having a brighter 28s band in gel. I have repeated about 30 times, with different criteria, but all in vain. Please help me :`(
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Hi,
I have 200++ SSR markers across genome, but I don't have enough resources to test all the SSRs on my samples. And I want to select SSRs that are distributed uniformly in each chromosome. How do I know which is the most suitable?
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S. Sodarsono's response is appropriate. If you have a genetic linkage map for your test organism available you can select the representative polymorphic SSRs for each LG or chromosome. As low as 14 SSR have been found to be discriminatory enough for genetic diversity studies in the past. Therefore, using 30 well distributed SSR across the genome of the test organism should be enough. However, if you intend to use them for mapping, you may need a lot more depending on genome size. This helped me long ago.
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I have found a Riccardia sp. in Himalayas with comparatively larger epidermal cells than the medullary cells (in stem section), however seems different from recently described R. elizabethae primarily due to lack of trigones in cells. Further, the epidermal cells shows brown pigmentation also. Can someone please inform about any other species with these character and suggest any reference about the same.
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Are the epidermal cells' wall of this species obviously thick? did you find it at a high altitude (about 3000 m)?
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Recently I tried to knock out a gene in rice using CRISPR CAS9. I got heterozygous plants with different mutations combination like (-3,-1) and (-6,-2) in T0 (I confirmed the genotype of T0 by TA cloning). After selfing the T0 transgenic plants (-3,-1), in T1 generation, I got only (-3, -3) homozygous plants. There were no heterozygous (-3,-1) or homozygous (-1.-1). The homozygous (-1.-1) may be lethal but I don’t understand how I could not get heterozygous plants (-3,-1). I completely lost (-1) mutation in T1 progeny. Any possible explanation for this???
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You could have had contamination with -1 DNA in your genotyping PCR and your plants were actually homozygous -3, -3. (most likely explanation)
Another possibility is that you have a mosaic mutation and the -1 mutation is only present in vegetative tissue. (possible, but more rare)
The -1 mutation could be a gametophyte lethal. Check the pollen with Alexander stain to look for male lethality. Reduced seed set will indicate female lethality. (very rare, but very interesting if true!)
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what is molecular pathway that is triggered under shade which boosts the plant height?
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Auxins!
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Which is the best protocol to extract RNA from grape leaves?
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Dear Karuppiah
The study at below showed that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from grapevine and many woody species.
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Dear friends,
Today I have got Atomic absorption data of my plant sample in which I have got one sample  showing 497.1 microgram/L means in PPB and the Blank reading is 16 PPB 
Now I would like to know the calculation formula of AAS data. The sample was solid material which was around 70 mg weighted and it was acid digested followed by made upto 50 ml final volume.
Blank was also processed same except material
Kindly refer me the exact formula to calculate the metal in plant sample with subtracting the blank.
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Metal weight mg/kg = (ppb concentration from AAS * 50)/plant sample weight
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I’m trying to extract RNA from anther of GMS  line of cotton. I’ve  used trazol method and modified CTAB but I didn’t get result.
Is there any suggestion?
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You may get best results from Spectrum™ Plant Total RNA Kit (Sigma). The isolated RNA may be directly used for RNA-seq study. The result is better than the Qiagen plant RNA extraction kit.
Best of luck!
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Hi folks,
I have a dataset where I'm exploring the changes in frequency and type of alleles within populations. I'm particularly interested in measuring allele frequency changes to rare alleles. Can any one suggest a particularly good metric or index of rarity?
Alternatively, can you recognize this index of rarity? A long time ago, I wrote down this equation but I *think* I may have written down the wrong author (Martin et al. 2004 is all that's scribbled in my notes). If you recognize the source of this equation, I'd be eternally greatful!
Have a great day,
LC
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There is a PLOS ONE paper that will be published soon, "An informational view of accession rarity and allele specificity in germplasm banks for management and conservation". It provides measures of "rarity" of populations and "specificity" of alleles, which is a metrics of their uniqueness.
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Begonias are sensitive to Oryzalin herbicide and it is not used for Begonia Polyploidy studies and i doubt if Begonia mini cutting can survive Oryzalin Treatments .
I want to use this article as base of treatments for my works 
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Hi
Oryzalin is a cheap anti-mitotic agent with low toxicity to other anti-mitotic agents, such as colchicine. I study polyploidy induction in Spinach and attached the article.
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I generated several transgenic Arabidopsis lines that contain a T-DNA in which two transgenes (Gene A and Gene B: see attached figure for more details) are linked. Each transgene has its own promoter and terminator. When I measured the relative expression of each transgene in the transgene lines, I found that the expression of each transgene was highly variable between lines which is probably due to the integration of the transgenic cassette in different genomic position. Most importantly, there was a significant difference between the expression of Gene A and Gene B in most of the lines whereas equal expression was found in one line. As both gene experience the same position effect,
1. Why was there a difference in expression between Gene A and Gene B. I guess this probably due to the difference in promoter strength. If this is due to promoter strength, then why in one line they are expressing equally?
2. Is there any other explanation besides those I mentioned above?
3. How should I test whether the difference in expression of Gene A and Gene B in the same line is a promoter effect?
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Differences in integration sites will put your cassette in a different regulatory context in each transgenic line. Since your Gene A and Gene B face in opposite directions, they can be differently regulated by local regulatory elements in the genome. Also, you can get methylation of your cassette after integration, which will down-regulate expression. Depending on the integration method, you can also get truncation of your cassette, which will reduce expression of just Gene A or just Gene B or both. Map and sequence your inserted cassettes to get a better explanation (if it matters). Or just screen more lines until you get several with the expression pattern you need for your project.
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I would like to request researchgate people to comment on the following abstract. Does this report any findings, or convey a message? What are the weakness of this abstract?
Improved design of a synthetic Bt gene stack and testing its insecticidal efficacy in the model plant Arabidopsis
Bacillus thuringiensis (Bt) insecticidal toxin protein encoded by Cry gene is a widely used technology to control insect pest in the crop field. However, development of insect resistant to Cry genes has appeared as a major threat to the durability of this approach, and thus urgent action is required to overcome this problem. Out of many available approaches, stacking of multiple Cry genes in the same plant is thought as the best strategy to delay the development of insect resistant to Cry genes. Here we report the insecticidal activity of a genetically engineered Bt gene stack consisting of Cry1B/Cry1C genes in the model plant Arabidopsis. Cry1B/Cry1C genes were designed to produce a novel version which is free of IP. Components which have the freedom to operate were used to test the insecticidal activity of the modified Cry1B/Cry1C gene stack. Availability of technology that does not require licensing agreement to use, is one of the main barrier to develop GM crops by public sector organizations in the developing countries. Thus, it is expected that the modified Cry1B/Cry1C gene stack will be a valuable tool to develop GM crops for public or humanitarian use in the developing nations.
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This abstract doesn't give any finding. it gives only suggestion about new methodology about gene transfering. the abstract needs key points about new methodology (why we are prefare, what will be happen when we use more than two Cry genes etc.)
it is still interesting subject. the review will get more attention.
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I am trying to identify and elucidate the function of a gene (let say X) in Brassica napus that control flower development in Arabidopsis. Using the sequence of Arabidopsis X, I conducted a phlyogenetic analysis based on protein sequence data. Phylogenetic analysis revealed that there is a single X in Brassica napus genome. After phylogenetic analysis, I isolated the cDNA sequence of X from Brassica napus by RT-PCR and then sequenced 20 randomly selected clone. When the coding sequences of these clones were compared, they revealed two unique sequence which were highly homologous except single nucleotide variation in some position of the coding sequence. When the two unique sequences of Brassica napus X were compared to its progenitor (Brassica rapa and Brassica oleracea X), I found that both of them are highly similar to the sequence of their progenitor X both at nucleotide and amino acid sequence level. The identity between Brassica napus X and Brassica rapa X were 98% (nucleotide level) and 93% (amino acid sequence). The similarity between Brassica napus X and Brassica oleracea X were 100% both at nuclotide and amino acid sequence level. My question is
1. What will be the possible explanation of presence of a single Arabidopsis X in Brassica npaus genome given the fact that Brassica napus is a tetraploid crop?
2. Are the two unique sequences of X found Brassica napus genome represent two different allele or two different genes?
3. I would also welcome any other explanation on this result.
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To really determine how many copies you have and if they are homologs, you will have to take one of two approaches.
1. Old school - Southern blot with Gene X as your probe. This will tell you how many copies of Gene X there are in the B. napus genome.
2. Bioinformatics - IF there is a high-quality genome sequence, you can look for the copies of Gene X as a prediction. Then, clone and sequence these regions from B. napus
Or combine both approaches. Good luck!
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Hello!
I would like to know the concentration of Basta that I need to use in transformed BY-2 cells in MS media, since I only find concentrations for seeds or whole plants. Can anyone help me?
Thank you very much!
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Hi Rita,
How did your BY-2 transformation work? Any success with the different concentrations that you tested?
Best regards,
Ward
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Plant Morphology versus Molecular phylogeny
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This question certainly requires specification. The answers depend heavily on what you are actually want to now.
(1) Morphological evidence considered in total evidence studies; A wide range and the selection depends mostly on the morphological disparity of the lineage inferred. Thus, the answer depends on the lineage of taxa inferred.
(2) Morphological evidence considered in the discussion of studies employing molecular phylogenetic: Again, the answer depends on the organisms studied.
(3) Material used to extract DNA for molecular phylogenetic studies: Again, the answer depends on the organisms studied and the question asked.
The hypothetical answers has one thing in common: it depends very much on the organisms studied.
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Can anyone kindly tell me what amount of rice seedling leaves and root is required for isolation of good quantity of RNA ? Should I weigh the freshly chopped samples before homogenizing with lN2 or after?
Also can anyone please suggest methods for storage of rice seedling root and shoot samples for later RNA isolation? 
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~100mg
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Do trees have an immune system? Is it crazy to consider immunizing trees against a fungal disease? I was brainstorming with a plant pathologist and we are trying to induce protection in trees.
If we had the genome and transcriptome of the fungal pathogen, could a vaccine be useful to "vaccinate" a tree of interest. We are talking about trees with high value, not forest plantation trees.
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Apparently, the concept of "vaccination" for trees has already been applied to, at least, dutch elm disease (see the link below). This is not "vaccination" stricto sensu, as pointed by Prerana, but recent findings about epigenetics and trained immunity in humans and CRISPR in bacteria indicate that pathogen recollection does not necessarily needs an adaptive immune system. So why not.
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Low RNA quality is a problem... According to literature, each cells might give 20-25 picogram RNA, assuming we extract 10,000 cells we must get something around 200 nanogram, while what we get is 2 nanogram. the problem is we need this RNA for further analysis like RNA sequencing, which is too low.
any suggestions about sample preparation, laser capturing setting and RNA extraction would be of high interest.
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Maybe the attached video publication can help you. It describes procedures for tissue preparation and Laser microdissection, also with regard to RNA extraction and quality control
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i am working on Gene expression but cant get it done because i am not getting any RNA extracted from my plants. my colleagues says its just because of rapid degradation. what are the main reasons for RNA degradation ?
i am using rice crop..
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Clean the area(where you are working), pipettes with 70% Alcohol and RNAse ZAP solution(I used Invitrogen). The eppendorf,tips,mortar pestle evrything you are using should be DEPC Treated then autoclave it( twice). During the RNA elution steps, carry out the process in ice so that the RNA donot get degraded. After you elute the RNA, perform the experiments in ice only. Keep in -80'c for storage and avoid repeated freeze thaw.
Check your RNA Quality in Gel and also in Nanodrop then proceed ....
All the best.
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Dear All, I am going to join Ph D in Biotechnology in india, i want to know some good research topics that can make my Ph D real good.My research interests are Molecular Biology, Virology, Agriculture Microbiology and Biotechnology, Plant Molecular Biology, Synthetic biology e.t.c any ideas pertaining to these fields are fine,
Thanks In Advance,
Sreerama M Mayur G
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You have a wide range of interests.  Pick an area/problem that really interests you and generates some passion.  Go to the websites of potential supervisors and read some of their publications.  Do they have a track record of successfully graduating PhD students?  Are they successful with grants (money is very important for equipment, lab consumables and attending conferences)?  Talk/email some of their former students or post-docs - what are they like to work with?  You are going to spend some time working with them so they need to be somebody that you can get along with.
But above all have fun and work hard!! Life is not a dress rehearsal and if something doesn't work out, cut your losses and look elsewhere.
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While hardening/acclimatizing micropropagaed orchids it is seen that lowering of the te,perature gives the best result. What might be the best suited physiological as well as molecular reason behind such a phenomenon?
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Recently was posted here, in RG, a review about the acclimation of an orchid genus, anyway you can download from this link
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picture is thymus vulgaris plant, that its leaves turn to something like chimera gradually. 
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Dear Dr Ghasem
please write me your  message in my email: ziarati.p@iaups.ac.ir
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centromere position in rice 1-12 chromosome
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You may refer to the genomic database of O. sativa.
and you'll find the exact position of each centromere here:
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I am trying to observe stomata by microscopy and want to measure stomatal aperture as well. My goal is to observe stomata opening and closing upon drought and cold stress on Pooideae grasses (e.g. Brachypodium).
1. Please suggest me some good literature for this method and tips on peeling (as the grasses are really difficult to peel). 2. How to measure stomatal aperture?
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I have tried the same steps as Sam. Very easy after the third sample.  Good luck
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Hi, I am aware that yeasts are eukaryotes and have the ability to do splicing such as in other eukaryotes. But I wonder, if let say, I want to express heterologous gene A in yeast, if I enter the whole native gene A sequence, how the yeast know where to cut--which are the introns and exons? On the other hand, if I insert only cDNA of gene A into yeast, would it be subjected to splicing--since they yeasts naturally do splicing, they may not know whether certain DNA has already been spliced or not? I hope someone could enlighten me on this as I am a newcomer in the field. Thank you in advance.
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As far as I recall expression of heterologous genes from genomic sequence in yeast leads to abherent splicing of introns/exons and is not recommended (someone with up to date knowledge of yeast expression systems may be able to correct me on this).  If you express a gene generated from cDNA none of the intron/exon boundary markers will be present anyway and these are highly conseved throughout eukaryotes so there shouldn't be a problem with further recombination.
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I am  optimizing protocol for avocado by Apical and auxiliary  bud culture. I initiated the shoot from both apical and auxiliary buds culture. I am currently on trial in optimizing the protocol for rooting of avocado. Can yo recommend and give me any citation regarding the hormone used for avocado rooting?
Thank you!
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Take a look at this document.
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Different methods are available for removal of selectable marker gene after transformation in the case of rice like co-transformation through the 2-vector system, super-binary vector system etc.
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Yes, that is what I heard that 'co-transformation through 2-vector system' approach is the simplest method to generate marker-free transgenic plants, although there are many other approaches, such as using site-specific recombination system (this is what we use). Vectors for 'Co-transformation through 2-vector system' are simple to construct. One vector contains your gene-of-interest, and the other vector contains the selectable marker gene.
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Hello every on, 
Pease I need the  protocol for the Dark treatments of seedlings of Arabidopsis. If anyine know how can I do that please help me .
Thank you
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I would like to add a general point to the above mentioned protocols.
For germination and subsequent growth experiments it is essential that the developmental programm of the seeds is insync. To acchieve this, I usually put the prepared plates (already wrapped in aluminium foil) for at least 48h to 4°C. Then, at the start of the experiment, I always put the plates for 4 hours to light (best in a light chamber for even light quality), wrap them again afterwards and put them to the growth chamber. The light pulse of 4 hours starts the germination program in all vernalized seeds at the same time. With this you minimize biological variation what results in smaller error bars.
Good luck with your experiments!
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I want to quantify the jasmonic acid (JA) content in plant using HPTLC. I am following the method described by Dhandhukia and Thakkar, 2008. J. of Chromatographic Science, 46:320-324. After the separation on the TLC plate, the JA band were not visible at 254 nm. I had also tried to stain the JA using Sulphuric acid as reported in some paper, but that also did not produce any band. What is the best option for staining JA on the TLC plate.
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Since jasmonic acid has low uv absorbance, treat jasmonic acid without fluorescence emission groups with fluorescence derivatization reagents
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from the plant micro RNA database,I found a microRNA sit-35 npr.This sit is the organism in which the miRNA is present, but I would like to know what is this suffix npr.Also I found some of the miRNAs with suffix akr.What is that
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Thanks for your reply
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Procedure of sterilization
30 sec ethanol
3 min HGCL2 %0.1 
5min sterilized water ×3
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I use 10% bleach with 0.1% Triton for my TC, 10 minutes (though this can be extended if the tissue is particularly tough) and 3-5 x sdw washes. Might be worth a try?