Science method

Plant Tissue Culture - Science method

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation.
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Which type of Microscope is best suitable for visualization of plant tissues and cell cultures and the organelles of cells and the same could be used for Karyotyping or for counting the number of chromosomes in cells. I want to use it in Plant Tissue culture Lab. Please guide me who are expert in microscopic studies.
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Dear Mr. Ali:
It is not necessary to buy the expensive microscope to do the plant tissue culture in the cell, tissue level. If so, you could match the computer with your microscope similar as for the chip manufacture. The most importance you need to note is of which the valuable plants to do those research.
David
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Dear Experts...
Your Suggestions are needed to make Plant Tissue Culture (MS) medium.
Could Ammonium Sulphate [(NH4)2SO4] be a substitute for Ammonium Nitrate (NH4NO3) in MS Medium?
If so..! what could be the amount per liter?
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All the best to you with many successes on your research
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I am looking for one that prints de result in the equipment by itself and also has the possibility to export all produced data to a computer.
Thank you in advance.
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Dear Dr. Mônica Bossardi Coelho,
The following info below may be good hint:
Spectrophotometer Instrumentation: Principle and Applications
By Biochemistry Den Posted on July 29, 2016May 5, 2022 Biochemical Techniques
_____
Spectrophotometer- Principle, Instrumentation, Applications
January 8, 2022 by Sagar Aryal
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From where we can procure Ammonium Nitrate (NH₄NO₃) for Plant Tissue Culture medium preparation?
Please suggest some leads
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For laboratory purpose you can can get from any Scientific chemicals company like Quality traders in Hyderabad.
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Good day, great minds.
I am a junior researcher working on some Nigerian plants that are not available here in Russia.
I have started a crazy though in my mind, about getting dried parts from the plants and try to regenerate them back. What is your thought on this? is there any better method apart from this on how to get the samples fresh in good health? Kindly share your expertise.
Thank you.
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If the cells of dried plant parts (leaves, stem, root, etc) are alive, these can be regenerated.
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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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After adding hormones(Auxin- NAA, and Cytokinin-BAP) to the culture media which contains agar and other micro, macronutrients as well as vitamins, Sucrose and Agar. On autoclaving at 1210 C for 15 minutes the media turned out to be coffee brown in color. Which should be slightly yellowish in color naturally. Is there any explanation for this? Does it have any faulty errors or contamination?
The snap is attached below!
Thanks in Advance !!
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Darkening of the medium is related to the burning of sucrose (caramelization), but I have never seen such a dark MS medium. Check the parameters of the autoclave, the concentration of each components and, as Ricardo Julian Licea-Moreno mentioned, quality of chemicals.
(NAA is auxin, BAP is cytokinin)
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SLOW GROWTH STORAGE for the mid-term conservation of a large number of species, including tropical and temperates. In this case, reducing the in vitro growth through the application low temperatures and less hours of light.
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The StarPac bags are sealed and held for 2 weeks in a growth room at 25 °C with a 16 h photoperiod provided by fluorescent lights (40 μM m-2·s-1). Then the StarPac bags are transferred at 4°C in low light. You can work with 12 to 16 h of photoperiod.
Best Regards,
Jean
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How to prepare Dicamba stock solution for initiation of callus in plant tissue culture?
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Ethanol and water combination. Use filter sterilized.
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A white structure is forming after 5-6 days of explant inoculation (without contamination), subsequently the material get contaminated. I tried 1 mg BAP, 2 mg BAP, 5mg BAP+0.5mg NAA etc., and the results were same. What may be the reason? will you suggest related publications?
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Subin Kunnath I don't know the species; however, since it's from India I must to assume that it's not a decidous tree, as walnut. Anyway, it doesn't matter. Bonga, during 80's, stated that a critical key to succeed with tissue culture of trees is to use juvenile explants: the higher the degree of juvenility, the higher the success probability. There're several ways to obtain juvenile explant sources. From the selection of the youngest branches to the grafting, passing through methods to force trees (or part of trees) to produce young branches. However, it doesn't assure the success, so an important part is your patience. Probably you'll need to introduce a lot of plant material before obtaining viable explants. Wish you success!!!!
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I am working on Plant tissue culture, recently my research plant showed green patches on the white callus and I had doubt that it may be somatic embryos. Therefore, I would like to know, is there any simple technique to visualise these somatic embryos under light microscope.
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First of all let them grow further, if you observe simultaneous shoot and root growth then they are SEs.
Then repeat the experiment as it will confirm that the results are reproducible, and that time try to observe the callus and globular, elongated globular or heart stage can be easily identified under light microscope without staining. If they are grown you will distinguish shoot and root (cotyledonary stage) which confirms that these are SEs.
As usually the growth is asynchronous, you can get different stages on one callus only but try to separate each stages under microscope and click good photo, it will be required for publication
Other observation is that they can be easily separated from callus, whereas shoots can't.
All the best.
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Hii, i need help, i have to come up with new idea using plant tissue culture, so i am thinking about producing a dwarf seedless rambutan tree. My idea is that to produce triploid plant which resulting in seedless fruit, then took the shoot meristem of tryploid plant and micrografting it to produce dwarf variety. Is this idea can be accepted?
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For the initiation of tissue culture of Philodendron pink Princess, I am curious about the starting material. There are some reports on different varieties of Philodendron concerning tissue culture from leaf explants, but couldn't see it for the pink princess. Have you ever tried tissue culture for this ornamental crop from its mature variegated leaves? Thanks.
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It is possible from old leaves.
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We know that somatic embryo can be used as an artificial seeds, but encapsulation include many kind of material. I ask to researcher to give their experience for us.
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Take a look at this video link
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Good day everybody.
I am looking for a good advice what stereomicroscope is capable to detect GFP fluorescent through the Petri dish. My objects are calli, plantlets, etc in vitro, so i want them to stay sterile inside the dishes with media. Once I tried to make samples (my explants destroyed) and watch them with fluorescent microscope but autofluorescence level was too high.
So, is there the easy way to detect marker gfp? What is the solution in your lab?
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We have used a NightSea adaptor that can be added to most stereoscopes. This option is much more cost effective if you have an existing stereoscope. We have used it to image calli. Here is the website- https://nightsea.com/products/stereomicroscope-fluorescence-adapter/ . You typically have to purchase them from EMS - https://www.emsdiasum.com/microscopy/products/magnifier/nightsea.aspx
We have success using this system.
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Using nanoparticles in PTC medium has positive results in in vitro flower induction, reducing bacterial contamination, somatic embryogenesis, etc., But what're the other technical challenges encountered using different nanoparticles in plant tissue culture? and What's the possible negative impact in the edible/ crop products?
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I think, one of the difficulties in using NPs is they form aggregates and this increases the size, than what we initially have. They could also accumulate in the plants and there is no way to see where they are localized and how much.
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Hello to everyone! I am trying to multiplicate a specific variety of Kiwi with tissue culture. I managed to multiplicate some. Some of them came from callus and some were produced directly from the starting material. What I would like to ask is, do the plants that come from callus differ genetically from the mother plant?
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The cytogenetic and genetic stability of the regenerated plants is one of the key prerequisites for efficient clonal propagation. Plants regenerated from relatively undifferentiated callus cultures possess a vast array of genetic changes. Such variations can result in useful agricultural and horticultural products. But sometimes, the variations in traits other than those of interest may be undesirable. Anyhow after regenerating plantlets, you can go for genetic fidelity assessment to understand the level of genetic variation.
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Dear Experts!
Silicon is used in plant tissue culture to enhance the growth and development of plants in vitro. I would like know the convincing physiological role of Calcium silicate (CaSiO3) in inducing totipotency/ Organogenesis/ Somatic embryogenesis?
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Yes, you are right Dr. Manokari, Silicon enhances the growth and development of in vitro shoots and roots via physiological processes only. It is still doubtful that it may induce Totipotency of plant cell.
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I only have 83% ethanol, but everyone seems to use 70% in other plant tissue culture labs. Is the 83% perfectly safe for the plants and my skin?
Diluting ethanol seems to be complicated. So it's better if I can directly use 83% instead of mixing distilled water to it. Please help!
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Hello Jay, properly diluting ethanol is very easy when you use the so-called "mix cross" which is unfortunately not as well known as it deserves. As shown in the attached diagram you write on the left side the concentrations of the starting solutions, in this case 83 (your 83% ethanol) and 0 (= water, 0% ethanol). In the center you write the target concentration (70%). Then you calculate the differences on the right side as illustrated in the diagram. The mix cross then shows you that you need to take 70 ml of your 83% ethanol and add 13 ml water to give 70% ethanol. It's that easy!
Good luck with your work and best wishes, Frank Edelmann
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Dear all,
Please inform me if there is any plant species that is challenge to plant tissue culture or micropropagation of that plant can never be done?
is there any such species of plant?
if it is there why and how it is not possible to culture those species?
thank you.
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According to the principle of totipotency, all plants can propagate under in vitro circumstance. Of course, according to the specifications of each plant (morphological and phsio-biochemical), there are always challenges. The whole effort of a researcher must be in making the final product cost-effective.
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Many pieces of researches have shown that responding explant percentage, embryo number, plant regeneration and also response rapidity increase in TCL explants compared to larger size explant. Now, I ask all researchers to share their experiences and reasons.
Thanks
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Minimal plant tissue offers many advantages over big explants. In case of TCL, cells are directly in contact with the nutrients, and have maximum chances of organogenesis and somatic embryogenesis on optimized growth regulators.
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I'm going to use half strength MS media for root initiation for indica rice cultivar. What will be it's composition when I'm using MS salt, sucrose and agar without growth regulators
Thanks in Advance
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Half strength of MS medium means the use of half the quantity of macro and micronutrients including sucrose. The gelling agent will remain in the same quantity.
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In some cases explants do not perform shoot multiplication or any other physiological responses, while others do under same growth/regeneration media. Think about the leaf explants in equal sizes which are capable of regeneration with a 80% of shoot regeneration. Why the rest do not perform similar response. How can we increase the percentage of explants forming shoot, and to which phenomenon(s) should we address that of high yield in certain explants?
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The nature of the explants is hidden in the source, the epigenetically different structure of the genome of the so-called meristematic or stem cells. In a developed leaf or stem, there are such cells only if it is a young leaf in the epidermal layer, capable of forming cells of stomata and their surroundings (rarely) and in cells surrounding the vessels, about the same in other organs, but somewhat more complicated. Questions arise because of the widespread "legend" about the totipotency of plant cells. In fact, just like in animal cells, the bulk of cells is characterized by the fact that it develops along the path of terminal differentiation. That is, it functions without dying until it is damaged irreversibly. Apparently, microautophagy was the solution to eliminate minor lesions, and to localize large autophagy and apoptosis of a separate fragment. At least we came to this conclusion after our research and their cytological analysis of various objects. Perhaps this should be discussed in the review so that the data does not disappear. If someone is interested, we will be glad to participate. In short - there is a meristematic cell - an explant is possible, if it is not - there is no explant.
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We know that light has a key role on different plant mechanisms, but its effect on the embryogenesis is question. Thanks for expressing the opinions of researchers.
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Yes, the nature of the color and its intensity can affect somatic embryogenesis experiments and may result in many differences among the obtained explants.
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I am looking for general MS media recipes. I can find hormone concentrations optimized for different species but I would like something more general purpose that can work for many species and may be optimized later. Can someone recommend MS media hormone concentrations for rooting, shoot multiplication, callus induction, and vegetative growth?
I have BAP, NAA, IBA, IAA, TDZ, GA3, 2,4-D, and kinetin to work with.
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Murashige and Skoog's media composition of macro, micronutrients, and vitamins. Hormones are not a part of MS medium. Even carbon source and gelling agent have to be added separately. You have to add the growth regulators according to your needs.
In general, cytokinins are required for culture induction, a combination of cytokinins and auxins for multiplication, auxins for rooting, 2,4-D to induce callus, etc. Proportion and types of growth regulators use to change.
Before starting your research, go through basic steps, plenty of information is available online.
Please go through the original article of MS medium
Article A Revised Medium for Rapid Growth and Bio Assays with Tobacc...
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Anyone wondered why and how Aloe vera leaves develop white spots.
(i) We’ve developed in vitro protocol for Aloe vera, and there were no white spots were appeared in the leaves when incubated under in vitro conditions.
Under in vitro conditions, white spots appeared,
(a) During delayed subculture
(b) When shoots are left with the minimal medium.
Similarly, the non-stripped shoots were developed white spots when transferred to ex vitro or in vivo conditions.
(ii) In the second experiment, the pups or offsprings (approx. 5 cm) developed from the mother plant’s root and rhizome, had no white spots. The offsprings were maintained indoor for 2 months and no white spots were observed. The plants developed white spots in the leaves when placed under shade as well as outdoor.
What is the mechanism behind the white spot development in the Aloe vera leaves?
Your valuable comments on the topic are much appreciated.
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Dear M. Manokari thanks for posting this interesting technical question on RG. As an inorganic chemists I'm absolutely no specialist in this field. However, I noticed that this seems to be a relatively common phenomenon. There are a number of closely related questions and discussions on the internet. For example, please have a look at the following potentially useful links:
Need help my Aloe Vera begun forming white spots
Why does my aloe vera plant have white spots?
White Spots On Aloe Plant: Reasons Why They Exists & How To Fix?
6 Tips To Fix Aloe Vera Plant White Spots Issue
Apparently such white spots on Aloe vera can be natural or caused by fungi. I hope this helps. Good luck with your work!
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How do you justify the usage of antibiotics in PTC at the time of
1) explant sterilization, or 2) within the plant tissue culture media.
is it suggestable/ethical to use antibiotics in normal tissue culture experiments? (not for the transgenic selection etc)
if yes how?
if not why?
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Isolate the contamination with loop, and check it under a light microscope. Sometimes it looks like bacteria by need eyes, but it might be yeast after a close look. Once you define the contamination type, apply an efficient chemicals. If it is bacteria, go through with its cell wall (- or +), then figure out the antibiotic and its concentration starting from 20 mg/L upto 100 mg/L. If it is not bacteria, go with an anti fungal agent depending on concentrations. Silver nitrate would be an effective chemical, but it reduces callus formations at high concentrations (>1 mg/L) in general. Good luck
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Expert suggestions are requested...!
In the Plant Tissue Culture, after few days (~10days) of subculture onto a fresh MS medium, plants which are growing in a glass jar, there was leaf defoliation observed, (browning of leaves, and falling on the medium).
How can I avoid this problem?
Need to add any extra additives/hormones to the medium?
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Dear Badhepuri Mahesh Kumar as M. Manokari has mentioned, sugar and N might cause in vitro defoliation. However, some other factors must also be considered. Microbial contaminants; the kind of vessel; volume of culture medium; explants inoculated per vessel; lenght of subculture; plants species; nutritive formulation; agar brand; kind and hormone concentration; among other factors, alone or combined can provoque in vitro defoliation. I hope it helps you.
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I want to make photographs of my plant tissue cultures for publications.
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Each and every stage and all the observations should be documented as photographs. find a good place for photography, it is an important step as it results depend on or are evidenced by the photos.
Adjust light sources to get good photos. Use a ruler while taking photos and finally you can draw scale bars according to the ruler sizes. If it is microscopic images, scale bars are available in the software itself.
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Dear Researchers, Greetings!
I am trying to develop in vitro propagation protocol for Dioscorea species and phenolic exudation was found at initiation and subsequent stages, but the major problem was at the initiation stage. I would like to know whether pre-treatment of explants to cold exposure at 4 oC along with antioxidants for few minutes can reduce the level of phenolic exudation in the medium. Suggestions appreciated.
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cold treatment although may temporarily slow down leach of phenolic compounds in medium but explant growth needs optimum temperature which being higher will not stop exudation of phenolics.
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Dear Plant Tissue Culture Experts,
Many crop and horticultural plants are produced by commercial tissue culture laboratories all over the world such as bananas, strawberries, sugarcane, etc. But many important medicinal plants could not be possibly propagated via tissue culture techniques at a mass scale, like Sandal, etc.
I want to know whether the commercial tissue culture of Rose is possible?
If yes, what are the protocol and PGRs? If not then why is it so?
Whether Rose has any different kinds of nutritional or physicochemical requirements under in vivo or in vitro conditions?
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Mahipal Shekhawat of course. A lab in Bulgaria, settled in the Rose Valley (Kazanlak), micropropagate commercially various rose accesions.
Here is the link
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Contacting plant researchers based at USA…
Dear All:
Would you please help us identify relevant research groups based in USA focused on the following areas? We are interested in research collaboration.
a) Plant tissue culture in temporary immersion bioreactors, and metabolites produced in vitro.
b) Plant tolerance to abiotic factors (e.g. drought and salinity) in the context of climatic change.
c) Characterization and (cryo-)conservation of plant genetic resources.
d) Plant genetic transformation and evaluation of side effects of transgenesis.
Thanks in advance.
Be safe.
José
--
Prof. Dr. José Carlos Lorenzo Feijoo
Head, Lab for Plant Breeding and Conservation of Genetic Resources,
Bioplant Centre,
University of Ciego de Avila, 69450,
Cuba.
Tel. 53 33 225768/212719
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Dear José Carlos Lorenzo Feijoo,
Plant scientists at the National Laboratory for Genetic Resources Preservation (USDA), in Fort Collins, Colorado.
Best Regards,
Jean
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Dear Researchers!
Since Activated Charcoal is an inert solid adsorbent material used in plant tissue culture to improve cell growth and development as well as adsorption of inhibitory substances in the culture medium. It is said that AC alters medium pH to an optimum level for morphogenesis How the addition of inert AC will change the pH of the nutrient medium?.
Thanks in advance...
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The change in pH results from the hydrolysis of sucrose, which could be increased by the presence of AC-
read the following contrasting works
Druart, P., De Wulf, O. Activated charcoal catalyses sucrose hydrolysis during autoclaving. Plant Cell Tiss Organ Cult 32, 97–99 (1993). https://doi.org/10.1007/BF00040122
and
Wann, S.R., Veazey, R.L. & Kaphammer, J. Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving. Plant Cell, Tissue and Organ Culture 50, 221–224 (1997). https://doi.org/10.1023/A:1005947008637
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Can anyone suggest me how to avoid fungal infection on regeneration media while doing agrobacterium mediated transformation of arabidopsis leaves by co culture method?
For agro removal i prefer to use cefotaxime and that works well but i immediately get fungal contamination hence no further callus initiation. This has happened several times with me in case of tobacco and arabidopsis both.
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I have had this in my explants as well. It looks like a similar fungi. I have found that it is in the soil of my plants and the spores must be on my explants and hard to kill with surface sterilization. My next plan is to empty the soil, wash the roots and transplant into fresh soil after I sterilize my plants. I also use PPM 1ml/L in my media to help reduce fungal contamination. Fungizone can also be used in small concentrations, but I have not researched that concentration yet.
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Sub culturing multiple shoots induced.
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The browning of the culture medium and the tissue is due to the production and oxidation of phenolic compounds from the damaged tissue. To reduce this problem, you can use antioxidant compounds such as citric acid, ascorbic acid, PVP or activated charcoal in culture medium
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In field studies, ethephon used as spraying agent, just to enhance growth, fruit ripening and flowering etc...
Actually i want to use Ethephon and triacontanol (plant growth regulator) in plant tissue culture. Is Ethephon and triacontanol used in liquid culture or suspension culture? or semisolid culture?
IF YES
Then How much concentration we can used ?
Any protocol / method ?
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Triacontanol increase the vegetative growth, chlorophyll content of plants, therefore, it is common to use it in the multiplication stage of PTC such as apples rootstock. Ethephon can be converted to ethylene in medium culture, but in prior studies, there is no significant effect on tissue culture.
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I have done genetic transformation of tobacco with leaf disc method. Now, my calluses have been sprouting small leaves. I want to confirm the presence of my transformed gene. Kindly suggest me, how long should I wait to shift them to shooting and rooting media? Or can I confirm the presence of gene at this stage without utilizing whole callus? Also, they have been catching fungus, so I am left with only few calluses. Shall I rely on them? or Shall I infect more explants?
Thank you.
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The attached article showing different steps of an efficient gene transformation method.
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Dear Colleagues,
I would like to ask the following question. In an experiment to reduce hyperhydricity in TIS bioreactors for plant tissue culture, I added 100 and 200 mg/l Phloroglucinol (Duchefa Biochemistry) to liquid medium. Unlike other (colourless) media, the medium with phloroglucinol always colored bright yellow after autoclaving. Although Phloroglucinol is described in the literature as autoclavable (e.g. Teixeira da Silva et al., 2013: "the anhydrous form melts only at 218-220 °C"), I wonder to what extent the degradation (resulting in a color change) of PG affects the efficacy of this compound?
Curious to hear about your experiences.
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Dear Reza Ghahremani culture media with PG just turn yellowish after autoclaving. This colour change seems not caused by sedimentation neither contamination. Other changes can be observed, as softening of gel.
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Does anyone have an innovative idea for cleaning the agar out of the corners of the Magenta boxes instead of jamming a brush in there? It takes a long time when you have to clean many.
Thank you, Tracy
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Soaking dishes in hot water with a little soap and sodium hypochlorite has good results. To prevent water stains, we use distilled water for rinsing.
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I'm trying to root in-vitro grown walnut, but the rooting percentage is extremely low. The plants were multiplied using DKW salts (Driver and Kuniyuki,1984) supplemented with 30g/L of sucrose, 4.4uM 6-benzyladenine and 0.05 uM IBA for 2 weeks under 16-h photo period. Then, i put the plants on root induction medium using DKW salts (Driver and Kuniyuki, 1984) supplemented with 40g/L of sucrose and 50uM K-IBA in the dark for 5 days. Following the root induction, shoots were put into fog chamber to root ex-vitro.
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Dear respected Anut,
The images you submited show that you have explant browning in your work. So my first recommendation is to control the production phenol and I suggest adding PVP in medium culture. Due to the tissue browning, it is better to use culture medium 1/2 or 1/4 MS. Also, In my experience, the best hormone to induce callus is TDZ.
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In tissue culture, it is recommended that most hormones be added to the medium by filter sterilization. However, when a pH value of 5.8 was added to a stock solution whose pH was not set to 5 and around, the pH value was changed by 1 point, sometimes more. I tried to adjust it with HCL and NAOH precisely and carefully but I could not succeed.
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If the hormone used is not heat sensitive, the best way is to add it to the culture medium, then adjust the pH and finally autoclave. If the hormone is heat sensitive, you can test the rate of change and adjust the pH accordingly before autoclaving.
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Non-destructive ways to measure (quantify) micropropagation "success" in woody plant tissue culture
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Optimal protocol in woody plants means the best performance at the tissue culture stage (establishment, multiplication, rooting). As we know, most of the growth parameters in these three phases do not need to destroy explants and plantlet, such as the number and length of roots and stems, the time of the first emergence of adventious roots, survival rate, responding explant percentage and etc.
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I try to get some haploid embryo by isolated microspore of Brassica napus. My donor plants are growing in two types of environmental conditions: chamber with total controlled environment, as also in a green house. My donor plants were in a good condition. Unfortunately in both case during in vitro culture I observed contamination. For buds sterilization I was using 70% alcohol for 3 min and 20% of commercial detergent + tween for 15 min. All surface sterilization was made very carefully. But the problems seem to like endogenous contamination, I am rather sure about this. How to avoid or destroy these annoying and disturbing microbes? I already use PPM (1ml/L).
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Dear respected Kamila Kozak-Stankiewicz,
From what you have said, it is understood that the in vitro and acclimatized plants were are contaminated, and this can be an indication of endogenous contamination. On the other hand, the use of alcohol alone can not have a complete effect on field mother-explants. I suggest you to use sodium hypochlorite (20% ) supplements with tween 20 as a surfactant and then washout three times with sterile distilled-water (3,5,2 min, respectively).
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Many teachers told us that it is hard to get some kind of species cultured by plant tissue culture, but why? What are the problems in plant tissue culture?
By the way, will it be meaningful if we try to expend the culturome of plant tissue culture now?
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Certainly, each method, despite its advantages, has its challenges. The history of using this method has shown that its benefits far outweigh its problems, and of course most of these problems have been solved. All the problems mentioned above are absolutely true, but the advantage of this method can not be ignored. The plant tissue culture method has been very helpful in preserving endangered plant germplasm as well as their genetic trasformation and plant physio-biochemical mechanisms identification. Other benefits of this method include: A) Callus induction; B) Embryo formation and proliferation; C) Embryo maturation; and D) Embryo germination E) Massive clonal propagation of genotypes of interest F) Production and propagation of disease-free plants by tissue culture G) Embryo rescue H) Management and modification of the ploidy levels I) Germplasm conservation J) secondery metaboilites production and etc.
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agriculture, soil, mycorrhizal fungi, biology
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If the volume of soil used is not large, an autoclave or micro-oven can be used for sterilization.
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Can turmeric be used as a substitute for the other comercially availble antifungal agents during plant tissue culture?
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I have not had the experience of using turmeric as an antimicrobial agent in PTC. In our country, this substance is much more expensive than conventional disinfectants. On the other hand, we know that turmeric has antimicrobial properties, but it is a natural phenolic compound and its negative effect on cell growth and proliferation has been reported. Also, in my opinion, since this yellow substance reduces the transparency of the culture medium and the environment becomes opaque, I preffer to use the transparent materials.
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A software which can help to design the experiment, store data, update new data, set subculture reminders, barcoding etc. Like Invitrosoft.
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I recommend Invitrosoft (IVS) software because you can use it in a tissue culture research with many option.
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Hi dear fellows
I am working on micropropagation of Begonia and Gloxinia which in both of them have internal bacterial contamination.
I tried ppm and antibiotics in media but both of them have negative effect on multiplication and growth of explants. As I am a mass producer of these two plants this contamination costs me a lot every year. do you have an idea that can help in controlling and eliminating endogenous bacterial contaminatin?
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In my experience, Silver nanoparticles is successful at 200 ppm concentration for 10 minutes.
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In my experience, Phytagel and Agar is more effective than others.
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Intensity of light required for plant tissue culture?
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As far as I know, 45 μmol m–2s–1 light is required for plant growth under in vitro circumstance.
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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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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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How can DMSO be used to obtain disease free material in plant tissue culture and sterilization of plant growth regulators?
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Dimethyl sulfoxide (DMSO) as an important polar aprotic solvent that used to resolve sensitive substance that reduce their capability under autoclave heating. Because DMSO is toxic to microorganisms, if it is added will sterilize the culture medium (after autoclaves).
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Among the several steps of plant tissue culture, the process of acclimatization is also very challenging because, if the in vitro produced plants can not be established in the field there is no meaning of investment in tissue culture targeting breeding.
Therefore, we are looking for a smart humidifier that can control the temperature and the humidity. When web searching we have found several kinds of humidifiers. but the selection of the one matching with the interest is difficult. so if someone has experience with it, would you mind suggesting the best one??
Thank you so much in advance.
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Do you need it for experiment or just for growing plants?
Actually, for acclimatization, plants don't really require very accurate humidity and temperature control. You only need an accurate one for experiment. It will also depend on your facility, e.g. how big your room is, whether air from outside can get in and affect the control of temperature and humidity, presence of cold area that could cause water condense, tolerance of other electrical apparatus to mist, etc. But in general, for tissue culture you can go with any type you can find. In fact, I would say buying separate thermostat and humidistat and connect them to normal heater and humidifier respectively would give you better control than buying a smart humidifier.
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I have been struggling with some plants in their in vitro propagation.
Can there be unusual methods in plant tissue culture? The other than the standard methods... Share me some citations please. Calling out to all Plant Tissue culture experts .
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Dear @Gaurav Mudgal
Tissue culture protocols may vary depending on plant species. You have not mentioned it. Why are you interested in "unusual" method? Are you working on variant method? If so, you may standardize it. If you are interested in references, use correct key words, and google; you will find so many articles and references.
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Hi everyone,
I will perform an experiment to evaluate the influence of MeJa in the production of secondary metabolites in root culture. I saw some articles and some methodologies, but I still confused about HOW the MeJa should be prepared correctly.
Have you ever done this before? If yes, can you please help me with this?
How much MeJa should I used for stock solution? How should I make its dilution?
Thank you! have a great day!
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The use of antibiotics, preservatives and similar chemicals is aimed to be avoided in cultures. Since isolations without the use of antibiotics and preventive chemicals is my goal, latent bacterial and sometimes fungal contamination is encountered. Does anybody has experience treating source plants with any preventive chemicals or antibiotics few days or weeks prior to isolation to reduce or eliminate the internal contaminants?
Any recommendations/suggestions will be highly appreciated.
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Just be sure to use sterile water for any rinses, no point in doing all the hard work to try and get surface sterile plants just to add back contaminants from non-sterile water.
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We often use 70-80% IPA or ethanol to wipe LAF and clean scissors and forceps... but how effective these cleanings are? How extant these IPA or Ethanol will help us to avoid contamination in plant tissue culture?
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Dear @Badhepuri Mahesh Kumar Contaminants in tissue culture laboratory are most commonly of biological nature, and can include bacteria, fungi, viruses and parasites. For each category, a separate disinfectant is required. For example, the work surface should be decontaminated with antifungal agent (5% Trigene) followed by 70-80% ethanol as antibacterial agents.
Although ethanol solution (70 or 75 %) is used widely as a disinfectant in cell culture room, the experiment or should be careful on the quantity sprayed and where to spray it, and particularly to avoid its contact with experimental cells, since this will lead to radical influence on cells pathophysiological condition.
Please also check a similar discussion at RG; hope, it may throw important insight for this discussion too:
Best wishes, AKC
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On the same medium composition i.e. 2mg/l 2,4-D and 0.4/0.5mg/l Kn MS medium(1962). It is found that leaf and petal explant gives embryogenic callus and node, internode and petiole derived explants with nonembryogenic callus.
In the same study I have performed one more experiment: node, internode and petiole derived callus were transferred to cytokinin rich medium for shoot regeneration; after one month shoot bud differentiation was observed in all the calluses (node, internode, and petoile derived callus) but there is significant difference in morphogenetic potentials of each explant derived callus. I have further subcultured each callus maintained separately and studied for 7 subcultures... during subculture on callus maintenance medium I tried the same composition. During each phase of subculture I have studied morphogenic potential of node, internode and petiole derived callus.. during this study I have found that petiole derived callus retain morphogenic potential up to six subcltures, while the other explants fail after 3rd and 4th subcluture... I need references for a similar study. If anybody has any suggestion please recommend some research papers on related work.
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The response of the cells present in different parts of a plant may vary in vitro. Because of the variable availability of PGRs in different organs, the explants respond differently to exogenously supplied PGRs under in vitro conditions.
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Is anyone used hydrogels instead of agar or gellangum as matrix for micropropgation/plant tissue culture experiments 
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We are using Plant Gel at the place of agar, it is a cost-effective and transparent gel and a very good alternative to the agar.
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Especially in Growth Chamber, we found same days culture some are good and some are attacked, Why ? and what are the Causes?
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Dear Abdul Main Although plant tissue and cell culture is performed in aseptic conditions, sometimes contamination occurs. Please go through the links given below; hope these could be helpful to you:
In addition, a similar question was put up earlier at RG; hope, answers to that related question might also be helpful to you:
Best wishes, AKC
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How can I wash it? Please suggest how.
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Water temperature is very important I agree with Mohiuddin Yatoo
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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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Good day. I am doing a study on plant tissue culture using fermented waste materials as a culture medium. I read some of the literature and most of the methods or procedures in preparing the culture medium are summarized. Do you know any reference materials with detailed procedures for preparing the plant tissue culture medium? Your help is very much appreciated. Thank you.
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از اطلاعات کلیهدوستان سپاسگزارم
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When you filter out or centrifuge down the cells during cell suspension culture protocol, is there any method to reassociate them back into one callus? What would happen if I just plated them all together in a clump (just like how it was if they had remained a callus)
Another question (which is unrelated to the first one), after the suspensed and separated cell are planted on the plate in the last step, what will happen to the growth of the cells if they happened to be touching each other (unconnected by plasmodesmata)?
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1. I am not sure I completely understood what you meant by "it's not a 100% yield right?"
2. The 3 statements you listed are all possible
3. <by putting in the antibiotic, you were able to select for only transformed cells.>
That is a textbook standard theory (the textbook would tell you so). But, In reality, you might get *chimeric* cell clumps, meaning a clump of cells constitute both transgenic and non-transgenic cells. This is might due to 'protection effect'. For some reasons, the transgenic cells can protect the neighboring non-transgenic cells from dying due to antibiotic selection.
4. Several things you don't want from your generated transgenic plant lines: (1) chimeric (not a homogeneous transgenic plant), (2) multiple copies of transgene (which might lead to gene silencing), and (3) sterile, such as no seeds (no progeny), (4) transgene can not transmitted into next generation
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I am wondering whether it is possible for two callus to be combined into one... both between genetically similar plants and genetically different plants.
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Yuan-Yeu Yau Take a look at the paper by O. Rosspopoff et al. (2017) that I attached in my reply to Settanan. (DOI: 10.1242/dev.142570)
Another important work is by Kareem et al. 2015 (10.1016/j.cub.2015.02.022)
And you might want to take a look at one on these review papers:
Sugimoto et al. 2019
and Ikeuchi et al. 2019
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Hi all,
Question as per above. I am undertaking some plant tissue culture at home and some protocols I have found call for 70% ethanol to disinfect explants.
Can isopropanol or methylated spirits be used or do the additives that make these things unpalatable also make them toxic to plants? I ask as these are readily available compared to 70% ethanol.
thanks in advance
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I have used Ethanol in dilution only. Spirits are dehydrating and can dessicate the plants. Usually bleach and Tween 20 are used for my plant samples. Be sure to add a sufficient amount of water for washes between washing with ethanol.
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In media preparation, the plant tissue culture media needs to be adjusted to ± pH 5.8.
Why it is so and what would happen if the pH is lower than 5 or more than 6.5?
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As mentioned already by the RG members following this thread, pH influences the availability and uptake of nutrients and plant roots' growth.
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I did tissue culture on guava plants in hopes of getting more quercetin compounds from these plants.
How to analyze the quercetin content in these plants? Can use HPLC-PDA analysis? How
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Dear Titus,
Please take a look to this manuscript https://doi.org/10.1155/2020/8853426
(It is open access). The authors did quantitative analysis of quercetin in different samples using an HPLC system
I have attached other papers on qualitative study of quercetin in in vitro plants.
Best regards,
Jean
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Fungal spores on surface of explants can be eradicated by surface sterilization with a fungicide. But, how to control endophytic fungal contamination form explants during plant tissue culture. Please suggest any chemical which can be added in tissue culture media or any method to control endophytic fungi from explants.
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I am in the process of trying to do tissue culture with Bucephalandra and/or Anubias; however, the resources on these two aquatic plants seem sparse. Is there a successful tissue culture protocol out there for these two plants?
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Dear Kara
These two articles could be beneficial.
Warms
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Thanks
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Could it be starting of callus tissue? Why it look white color?
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Thank you Dr. M. Çağrı Oğuz and Dr. M.K. Tripathi for your attention
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I'm looking for research in the plant tissue culture area for my undergraduate research. And I m thinking of developing it as a business in the future. So please suggest a topic?
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By propagating ornamental plants
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Good day. I am doing a study on plant tissue culture using fermented waste materials as a culture medium. I read some of the literature and most of the methods or procedures in preparing the culture medium are summarized. Do you know any reference materials with detailed procedures for preparing the plant tissue culture medium? Your help is very much appreciated. Thank you.
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Good day. To prepare the culture medium you need to start by deciding on the concentration of your stock solutions for your chosen medium. Most researchers usually prepare four components (stocks) of the culture medium: which are macro elements, micro elements, vitamins and chelate (iron stock). In most cases preferred strengths of the highly concentrated stocks are 20x for macro elements, and 200x for macro elements, vitamin stock and a chelate stock. The dilutions are made from these concentrated stocks as follows, for a 1litre culture medium: take 50ml of the 20x macro element stock and 5ml from the other 200x stocks (micro, vitamin and chelate). This is basically how the media are made. The other components are made and added after having made these stock dilutions. These components are usually hormones, sugar sources like maltose, sucrose etc, solidifying agents like gelrite or phytagel, casein hydrolysate, and so forth. Some hormones are heat labile and are added after autoclaving the medium, one needs to know how stable are the hormones when exposed to high temperatures. The pH of the medium is adjusted using 1M NaOH or HCl before autoclaving. The medium is autoclaved at 121 degrees celcius for 20 mins, and after cooling down an amino acid L-glutamine is added before dispensing the medium in a sterile laminar flow bench. My humble apologies that I do not have a reference material as per your request however feel this summary will help you make your medium without much difficulty. This is a detailed plant tissue culture medium preparation procedure.
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