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Plant Regeneration - Science topic

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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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I would suggest you study the following article
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Various plant growth regulators were found to have a positive effect on organogenesis and somatic embryogenesis, but which one is more important?
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Dear @Reza Ghahremani
Different plant species, such as C. canephora, A. thaliana, and Musa spp. responded successfully to the Somatic Embryogenesis induction using different explants, conditions, and concentrations of PGR. The details can be accessed at:
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What are the roles of Mannitol, Polyethylene glycol ( which one 4000/6000/8000) and Absicisic acid in somatic embryogenesis in callus for plant regeneration?
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Dear Swati,
Please take a look at these 2 interesting papers attached. I hope it will be useful for the discussion.
Polyethylene glycol and abscisic acid improve maturation and regeneration of Panax ginseng somatic embryos
Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos
Best regards,
Jean
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Many protocols exist for plant regeneration through axillary bud/ apical bud developed in the rhizome.
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we normally use Ck and auxin,(or 2iP) but it may be very basic
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Although it's not a very effective sterilizer alone for plants but still being part of treatments in all researches. i wonder over its widely essential use and what factors decide the time of ethanol treatment?
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Dear @Amina Ilyas You may find a detailed discussion related to your query at the link given below as to why 70% ethyl alcohol is used for surface sterilization:
I would like to add that I am fully convinced with the explanation by @Yuan-Yeu Yau on that thread.
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Since tween 20 is no doubt an excellent surfactant to remove surface tension and provide better exposure of plant surface for sterilization, by removing the bubbles which are more problematic in case of plants having plenty of hairs and trichomes. I wonder if in case of unavailability of tween 20, can a common detergent or dish soap provides the same benefit or there is any other cheaper compound as a replacement?
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Tween 80. Maybe Triton-X-100. Especially Tween 80
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1.Technologie des Légumes (Y. Tirilly - C. Bourgeois) Tec Doc 1999 - ISSN : 0243-5624 : Génétique et création de nouvelles variétés de légumes (M. Branchard - M. Pitrat) 27-44.
2. Variability in plants regenerated from tissue culture (E. Earle - Y. Demarly) Praeger1982- ISBN : 0-03-059364-6 :In vitro culture of barley: a method to study Rhynchosporium scald disease and select plants resistant to the toxin, rhynchosporoside (M. Branchard) 343-350.
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Michel Branchard you can scan the two papers into electronic files first. Then you upload the files into ResearchGate.
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I am doing Agrobacterium-mediated soybean hairy root transformation. I want to know which phytohormone performs better for plant regeneration in it.
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For hairy root transformation after co-cultivation you just go for MS+2,4 D (0.5 mg/ml) in less concentration or u can use IAA in 2-4 mg/ml.
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In my career in plant tissue culture and biotech research, I encountered, more than once, a situation in which I could not repeat results described in peer-reviewed articles. This is a very disturbing and disappointing situation. Scientific progress is hampered if findings of a study can not be repeated. I wonder whether other scientists met the same kind of problem? What was the type of difficulty (e.g. inability to obtain plant regeneration or plant transformation)? What do you think is/are the main reason/s for irreproducible results?
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Very good and true remark! Working in biotechnologies I’ve often encountered the same problem, i.e. the impossibility of obtaining reproducible results of my own experiments, carried on by the same people, keeping strictly the same process conditions and quantities of materials taken with high precision. Furthermore, duplicate or triplicate samples in simultaneous experiments give very different results. This has happened to me since I work with biochemical, fermentation processes, involving living matter, bacteria, plant and animal biomass, active sludge. For comparison, when I worked with chemical processes (synthesis), the repetability and reproducibility of the results was very high. My explanation is that the biochemical processes are much more complex, dynamic, rather unpredictable, and the biological factor is responsible for the poor reproducibility. Bacteria and living matter generally work by complex mechanisms that involve very different biochemical pathways, specific enzyme and high instability even for the same conversion process. Often it is not possible to know exactly which product will be obtained, on what biochemical pathway, under what environmental conditions and at what time in two identical experiments involving bacterial metabolism. Although as a whole, even in detail, science has made tremendous progress, it seems that living matter has its strict information that does not allow us to fully control it. This is an explanation in the lack of explanation...
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What is the impact of AGL1 Agrobacterium strains on regenerated plant phenotypes? AGL1 is hypervirulent. I also heard that some plants regenerated from calli are erratic. Are there any papers claimimg so? If you could find me a paper stating the same.
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A lot of researchers have used AGL1 for genetic transformation on different plant species. Plants regenerated from calli with erratic phenotype are not necessary due to the Agrobacterium per se. The erratic phenotype or regenerants could be raised from tissue culturing practicing, or due to somaclonal variation.
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Hello everybody, I am working in a conifer tree species, initially i need to propagate the plant through callus, i have established the callus induction but i struggle to regenerate the plant through callus, so i have planned to regenerate the plant through epigenetic remodeling. can we regenerate the plant through DNA methylation process..? i looked at the protocol for this. i would appreciate if anyone have experience in the plant regeneration via epigenetics. Thank you.
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You may find useful these references:
However, epigenetic remodelling is not something you can manipulate at will, it depends on the cell developmental state, the tissue type, the environmental conditions, and the hormonal stimuli present.
My advice is look for available regeneration protocols for close relatives of the species you want to regenerate and test how these work on your case.
Most trees, including conifers, have extremely large genomes with high duplication and tend to have a large number of transposable elements of various classes. It may be hard to promote plant embryogenesis from callus, and sometimes direct organogenesis is more effective.
An example:
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I am working on transgenic rice. We have standarised the regeneration and transformation protocol for it. But I could see two Albino plants that are regenerated from calli. Can you please help me to short out the issue. For your kind information I want to mention that other plants regenerted with same media and hormone componets including same light condition have not shown such abnormalities. Please find the attached file.
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The transformed rice seeds having kanamycin resistant gene as selection marker can produce albino plant in vitro. This is not unusual as kanamycin at particular concentration blocks ribosomal RNA synthesis in plastids resulting in no synthesis of chlorophyll.
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Hello, I just started a project trying to produce eucalyptus protoplast and we wish to regenerate plants from it. Can someone give any advice what is the best material to start protoplast production to have successful plant regeneration from it? Is there any protocol that you could share? Thank you!
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I am using tobacco callus for suspension culture in MS basal and its color is brown. I did not find any contamination in suspension when plated in nutrient agar. callus was friable and white in color.  
can anyone give me some paper on plant regeneration from suspension culture.   
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Are those cells dead?? You said you cultured it in MS "basal' medium. Does this MS basal powder you used to make medium contains other important ingredients such as vitamins...etc? There are many types of MS powder mix sold from companies. Some contains only macro-elements, some contains both macro-elements and vitamins,......etc.
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Dear Researchers,
    Has anybody tried protoplast culture and got plant regeneration from that? I want to try it in cotton if possible. Experts please share your experiences and ideas. Thanks in advance.
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Yes, I have done that on tobacco. See attached paper. Pictures of the results from different steps are shown in p.160 of the paper. However, I have not tried that on cotton. You can use the paper as your reference.
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Some time we study whether a particular stand of trees produce sufficient regeneration or it must be supplemented through artificial regeneration. In this case regeneration of the stand is studied where number of seedlings is counted. But it not clear to me that up to what diameter of seedling may included in the study? Please guide me
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Thanks Andrew
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I am interested in any experiences in anther culture and/or isolated microspore culture - media, pretreatments of the flower buds, plant regeneration efficiency. In addition, is there any reliable method to distinguish homozygote regenerants from the heterozygotes in pea?
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plant regeneration efficiency. Furthermore, is there any reliable method to distinguish homozygote regenerants from the heterozygotes in pea.
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Not aware of anything from the Ghats (not my region) but these may help:
Balch, J.K., D.C. Nepstad, L.M. Curran, P.M. Brando, O. Portelab, P. Guilhermeg, J.D. Reuning-Scherera, and O. de Carvalho Jr. 2011. Size, species, and fire behaviour predict tree and liana mortality from experimental burns in the Brazilian Amazon. Forest Ecology and Management 261: 68–77.
Cochrane, M.A. 2003. Fire science for rainforests. Nature 421: 913-919.
Cochrane, M.A., and M.D. Schulze. 1999. Fire as a recurrent event in tropical forests of the eastern Amazon: effects on forest structure, biomass, and species composition. Biotropica 31: 2–16.
Kinnaird, M.F., and T.G. O’Brien. 1998. Ecological effects of wildfire on lowland rainforest in Sumatra. Conservation Biology 12: 954-956.
Uhl, C., and J.B. Kauffman. 1990. Deforestation, fire susceptibility, and potential tree responses to fire in the eastern Amazon. Ecology 71: 437–449.
I would be keen to hear if you find anything more relevant.
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Which salt can be used as alternative to Ammonium nitrate in callus regeneration media?
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Dear Dr. Kaushal
You can use Potassium nitrate or Ammonium sulfate for callus regeneration.
Thank You.
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Hi,
I am initiating callus from hypocotyl in Catharanthus roseus, using MS media with 1mg/l of both NAA and BAP. The callus looks good. My concern is that the color of the callus is dark green and although it is not generating any shoots or roots at the moment (after about 20 days), I am afraid it will start to regenerate. I don't want that and I need to keep them as they are. Should I make any changes? Is it normal for Catharanthus roseus to have dark green callus? 
Any thought or suggestion is appreciated.
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To prevent green coloration you may change the photoperiod (increase the dark cycle). Variations in NAA/BA combinations may also provide desired results.
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I want to know if the whole plant regeneration is possible from root
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Dear Hawer,
From root explant follow organogenesis protocol in tissue culture then you can get some callus and from there you can able to get plantlets. If you will clear about genus name it will be easy to help you.
Regards,
Aloka
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Somatic embryogenesis for in vitro plant regeneration provides several advantages over traditional organogenesis. However, some researchers believe that somatic embryos have less genetic stability during micropropagation. Is it true? Why?
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Yes, somatic embryos have less genetic variability compared with the regenerants from calluses. Somaclonal variation depends upon the concentration of auxin particularly 2,4-D. If plants are not produced from a callus after 4-6 months, attempts should not be made to regenerate plants and new cultures should be initiated.
Please read this paper:
Singh, R. J., T. M. Klein, C. J. Mauvais, S. Knowlton, T. Hymowitz, and C. M. Kostow. 1998. Cytological characterization of the transgenic soybean. Theor. Appl. Genet. 96: 319 - 324.
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Including which projects of P. euphratica for plantations & restoration were successful or not, and what were the outcomes in terms of habitat restoration. If P. euphratica is introducing new area, what will be a potential ecological hazards?
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Three good textbooks about plantations, mainly in the tropics, but they do not refer to Populus euphratica:-
Evans, J. 1992. Plantation forestry in the tropics. 2nd edition. Clarendon Press, Oxford, UK. 403 p.
Garforth, M. and Mayers, J. (Editors). 2005. Plantations, privatization, poverty and power. Earthscan Forestry Library, London, UK. 294 p.
Susuki, K. et al. (Editors). 2006. Plantation technology in tropical forest science. Springer, Tokyo, Japan. 292 p.
In the late last century and early 2000s major reports on plantations were produced by FAO, Rome, Italy, especially authored by Jim Ball and Jim Carlyle. These may be available online. The Forest Research institute in Izmit, Turkey (formerly the Poplar research Institute) may have written on P euphratica.
I hope this helps somewhat.
Regards, Jeff Burley
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my plants regenerated in vitro are suddenly browning
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The plant in the picture is undergoing necrosis. Necrosis can sometimes occur spontaneously in vitro as a consequence of stress (sometimes even transferring your plants to fresh medium can cause stress), but it can be also due to medium composition; please check if the composition of your cultivation medium is appropriate, also check its other properties, like pH.
If this is the first time that you're trying to cultivate that plant in vitro, you should look for optimization of the medium composition. If you have done that before and had no necrosis, then just forget about this plant and look for healthy, viable explants.
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My question is in context of looking for plant regeneration post fire. Also, how are seedlings and saplings as recruits categorized? Papers I referred mentioned different sizes to define these categories. I am also interested in knowing what are the hiccups researchers faced while monitoring fire in deciduous and grassland ecosystems. 
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When studying prescribed fire, you have the added benefit of establishing permanent plots prior to the burn which you can sample both before and repeatedly after the fire. Pre-burn conditions that can be important to vegetation recovery and tree regeneration include the presence of plant species that can recover vegetatively (a strong determinant of what comes back!), depth of the litter layer (if any, which can determine local mortality due to fire intensity or smoldering), and local crown closure or canopy cover (e.g., measured using hemispherical photography or a spherical densiometer). I hadn't thought of sampling the seed bank, as suggested by Mathiventhan, but that is a good idea and could be important in some systems too.
My experience is in the Pinus contorta forests of western Canada, where vegetation responses and forest recovery can be very patchy, especially under a mixed fire regime or the relatively cool conditions that managers are comfortable with for a prescribed burn. So make sure you have lots of sample plots and be prepared to stratify your results by pre-existing vegetation (you mention a forest-grassland mosaic) and local burn severity.
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Yes...What do you want? some hairy roots really sprout shoots. I dont care, as I can use the shoots for transformation. But if one is only after those shoots, are they transformed too and perhaps root easier later on?
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The name of my species is Symplocos recemosa Roxb.,which is rare engendered and threatened medicinal species .There is no seed germination possible in vitro as well as ex vitro as the seed coat is very hard. Now I am using nodal explants, shoot tips for vegetative propagation of the plant. Nodal culture gives not more shoots, as I am aiming for mass propagation of the species. What is the best explant to regenerate for getting maximum shoots?
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I would suggest callusing in vitro using cytokinins and auxins with subsequent transfer of obtained calli into the fresh liquid medium in e.g. bioreactor systems such as temporary immersion ones.
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Callus induction and plant regeneration studies by manipulating plant growth regulators.
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