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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What is the best medium for the rapid multiplication of bamboo plantlets by micro-propagation ? Are there any rules of thumb in using ingredients including cytokinin, auxin and others that could promote rapid shooting and rooting? Appreciate if those who identified any tricks could be provided.
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Liquid MS Medium
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I am facing lot of bacterial and fungal contamination during initiation stage of bamboo tissue culture. we are using tween 20 , 70% alcohol and hgcl2 for surface sterilization.
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Thanks for your help.
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Sorry to bump old question but this has become a serious issue for me today so sharing
If your plant species can't handle much chlorine you can use dilute phosphoric acid to adjust downward. If it also can't handle much phosphorus, use dilute acetic acid
For upwards adjustment most people use dilute KOH, because a lot of species are very sensitive to sodium. Where that adds too much potassium, you can also use very dilute aqueous ammonia
Everything dropwise, with checking inbetween. I'm finding some media recipes I've been working with have strange buffering properties. You don't want to be backward and forwarding beyond the recommended pH too often or you'll really throw off the recipe's specificity and precision
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There is statement of Callus producing less secondary metabolite as it is undifferentiated cell. Does differentiated tissue like shoot/root from plant tissue culture produce more yield of secondary metabolite.
Why does differentiated produce more than undifferentiated cells.
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Yes, differentiated tissues like shoots and roots from plant tissue cultures can generally produce higher yields of secondary metabolites compared to callus, which is undifferentiated tissue. This difference in metabolite production largely stems from the unique structure and functionality of differentiated cells, which is absent in undifferentiated callus tissue. Here’s a breakdown of why differentiated tissues tend to yield more secondary metabolites:
  1. Specialized Cellular Structures: Differentiated tissues such as roots, shoots, or leaves have specialized cell structures that are directly involved in secondary metabolite synthesis. For instance, glandular trichomes, which are present on the surface of leaves or stems, often contain high concentrations of essential oils and other secondary metabolites. These structures are absent in callus tissue.
  2. Enzyme Pathway Localization: Secondary metabolite synthesis often involves complex biosynthetic pathways that are compartmentalized in specific organelles (e.g., vacuoles, plastids) or within specialized cells in differentiated tissues. Callus tissue lacks this cellular organization, so it cannot efficiently localize or accumulate certain metabolites.
  3. Differential Gene Expression: Genes encoding enzymes responsible for secondary metabolite biosynthesis are often more actively expressed in differentiated tissues due to tissue-specific regulatory factors. Callus tissue may lack the necessary signals or regulatory mechanisms to trigger these pathways effectively.
  4. Environmental and Hormonal Responsiveness: Differentiated tissues respond more robustly to environmental signals or phytohormones, which can further enhance secondary metabolite production. In vitro culture systems can use this by exposing shoots or roots to elicitors (e.g., jasmonic acid, UV light) to stimulate metabolite production.
Due to these factors, culturing differentiated tissues or even using organ cultures (e.g., root or shoot cultures) is often a better strategy for enhancing the yield of secondary metabolites in plant tissue culture setups.
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Understanding copper sulphate decomposes at high temperature, can I supplement it to the media before autoclaving or should it be after autoclaving the media?
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The answer making by Raghad Mouhamad, our great colleage is perfect, because he explain step by step with very good reasons.
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We use Himedia PCT grade Clerigar for plant tissue culture in our lab.
What are the ways we can dispose this gel after cultivation period?
When stored for disposal, the gel liquifies and ruins the package. Is there any way to solidify this used clerigel?
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Hemalatha Rajendran Disposing of used Clerigar gel properly is essential for maintaining lab safety and environmental responsibility. One effective method is to solidify the gel before disposal. You can achieve this by storing the used gel at lower temperatures, such as in a refrigerator or freezer, which may help maintain its form and prevent leakage. Additionally, mixing the liquefied gel with absorbent materials like sawdust, cat litter, or other biodegradable substances can facilitate solidification, making it easier to handle and dispose of.
If your lab has access to incineration facilities, used gel can be disposed of this way, provided it complies with local regulations regarding waste disposal. Once solidified, the gel can also be disposed of in regular waste, assuming it is not classified as hazardous. It’s important to check local guidelines to ensure compliance. Alternatively, you might explore the use of commercially available solidifying agents that can be mixed with the gel to transform it into a more manageable solid form.
Lastly, some laboratories have waste management programs that accept certain types of gels for recycling or repurposing, so it’s worth investigating if that option is available to you. Always remember to consult your institution's waste disposal guidelines and adhere to any specific regulations or protocols for hazardous materials.
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To combat environmental contaminants, plant tissue culture labs are fumigated. Would vapourised hydrogen peoxide have a negative effect on the tissue cultured plants in jars?
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In plant tissue culture labs, vaporized hydrogen peroxide can be utilized for sterilization. It's a non-toxic, efficient way to clean surfaces, tools, and even entire rooms. This method offers complete coverage in difficult-to-reach places and is especially helpful for materials that are sensitive to heat. To guarantee efficient sterilization, however, without harming plant tissues or equipment, the right concentration, exposure duration, and safety precautions must be taken.
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Does anyone have experience with plant meristem culture? What kind of plant growth regulator combination is best?
From what I read, there seem to be 2 methods. One using cytokinin (BAP, kinetin) combined with auxin (NAA, IBA) that reminiscing shoot cluster generation from leaves or callus. I presume that is also what being attempted here, turning the meristem tissue into callus and then to shoot cluster. Another method though uses cytokinin (BAP, kinetin) combined with gibberellin (GA3), which I presume is to maintain and elongate the meristem back into a single shoot tip instead of turning it into a shoot cluster.
What are your experiences with any of these methods? Which one worked for you, or do you have a different method?
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No method will work for all. that is where you had to research your media composition and phytohormones which suitable to genotype you are working with.
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I want to determine total flavonoids from plants samples but I am afraid I might not have enough dry sample (weight) for proper determination of flavonoids. is less than 100 mg enough for extraction?
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It is possible to determine TFC in this case. However, lower mass can increase possibility of error. If this is my sample I will dissolve it in 1 mL of some MeOH or EtOH and use extract for further determination.
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We submitted a sample of rice root for the purpose of quantifying phytohormones. Upon receiving the results, it was found that the sample contained BAP, a synthesized phytohormone that is typically supplied to plant tissue culture media. Can we detect and quantify 6BAP or BA?
Thank you
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Thank you very much Raghad Mouhamad
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If you are interested in collaborating project on Biotechnology, Bionanotechnology, Microbiology, Plant Tissue Culture etc., Please let us know.
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I am honored to participate with you in research. Please kindly explain the ideas and plan.
with all my respect.
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what are the components contain for Plant tissue culture ? How can it prepare for 1L solution of media for Plant tissue culture?
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Hi,
It does depend on the purpose of the culturing and to some degree the species. There is unfortunately no "one size fits all" but generally MS media is made at 4.4 grams per L. You can add it to the solution with your agar (check that your pH is correct- usually in the pH7 range but again depends on what you are growing) and autoclave. If you need to supplement with hormones,vitamins, antibiotics, or some carbohydrates you add these after it has been autoclaved using 0.22 uM filters.
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Counts data re-distribution.
I ran ANOVA however, the numbers don't make sense. My experiment was based on treating plant tissue culture explants with NaDCC and PPM. After a 30-day period, I had to count the number of aseptic and contaminated tubes from a batch of 10. So if 5 tubes were contaminated it means 50% were aseptic. On the other batches, I had O%-contaminated tubes and 100% clean tubes. Therefore, there's a huge variation between the numbers resulting in my data having a non-normal distribution or skewed distribution. For me to use ANOVA I assume my data must be normally distributed. The tubes were counted and values were converted to percentages. But when plotting graphs in Excel +/-SD extended passed zero to the negative quadrant. I am not familiar with statistics or ways to code, I am completely clueless on how to resolve this issue.
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yes for non normal data we use non parametric statistics, alternative to ANOVA is Kruskal Wallis, equally performance as ANOVA
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Hello,
I'm working on plant tissue culture. Sometimes the tissue I cultivate produces what you can see in the photos attached.
I'm wondering if these are also calluses, or they can be organ regeneration?
Thanks in advance for your precious comments
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Hi, it is difficult to predict your results and where you have to go. For plants regenereration through organogenesis or somatic embryogenesis you have to be certain according your objectives of your research. Should be to explain better?
Thank yoiu
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Hi everyone. Is the use of Phytagel better than agar in Plant tissue culture? I work with stevia and sugarcane, does anybody have experience in these crops in vitro? Or does anybody prefer the use of agar instead of Phytagel at any other crop? Why?
Thanks in advance.
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Well, it seems to depend on the species and cultivar you work with. I like Phytagel, in part because it allows me to see the roots. In my experience, Phytagel is a more consistent product than the range of agars.
As tissue culture tends to be pricey, folks often don't change up the medium unless it is needed for optimization.
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Hello dear researchers,
Considering some research on plant tissue culture and induction of callus in order to evaluate and compare the production of a secondary metabolite in the callus with that in the plant in the field,
1) How do you determine how long the experiment and the callus sub-culturing should be continued so that you can come to some valuable conclusions?
2) How do you determine in which increments the samples of the callus and the media should be taken for evaluation of the growth of the callus and the amount of metabolite production?
Thanks in advance for your help
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1. Normally we do subculture after 30 days. But there are some plants rich in phenolics, they turn brown so you can reduce the frame. But it mainly depends on the initiation of the shoot tips. You should specify the plant name so suggestions can be more specific.
2. Regarding callus I would suggest take 1g and take liquid media 30 ml in a 50 ml conical flask sealed with stopper. Keep a look on contamination and transfer after 15 days by taking inoculum into a fresh media.
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I am working with a nitrogen sensitive species trying to induce somatic embryogenesis. The textbooks I've gone through so far insist that casein hydrolysate - or another reduced nitrogen source - is crucial to initiating embryogenic callus. but many of the protocols I've read don't include CHL or any other nitrogen sources. So what's the truth here? Is this reagent crucial or not? I've ordered the casein hydrolysate just in case, but am trying to figure out if I'll even need it.
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Regarding the omission of nitrogen sources I think it may be ammonium ions which is initially omitted in many research during the microcalli development and casein hydrolysate on the other hand is mixture of amino acid. But organogenesis is dependent on many factors like carbon source or hormones or additives so you have to standardize it. You can order MS without Ammonium salt it is available in duchefa.
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We'been preparing B5 media for plant cell culture (MS + Gamborg vit+ 3% sucrose) with and without agar, adjusting with KOH to pH 5.7. We noticed that the pH changes after autoclaving, around 0.7 units, so we have to readjust..
Has anyone had this problem? any suggestions? Thanks
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Haven't specifically checked this or used B5 but for our normal MS medium recipe we use MES as buffer, maybe this could help?
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Normally, we use oxidative agents like hyperchlorite to sterilize plant tissue to introduce into micropropagation. However, we are doing a plant that has high phenolic contents, and the oxidative agents caused severe browning. Is there any substitute disinfectants that is not of oxidative type to use in this case? We can't use mercury though because of its toxicity.
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Dear Duy Minh Pham I used ascorbic acid for date palm once and it was successful. depending on what explant you use the concentration and duration of treatment will be different. according to my experience the ascorbic acid only prevent browning during the sterilization but not at the initiation stage.
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Plant tissue culture: ammonium sulfate instead of ammonium nitrate? what concentration? how to prepare stock 1 solution for MS media using ammonium sulfate?
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Ammonium sulfate can be used as a source of nitrogen in plant tissue culture media instead of ammonium nitrate. The concentration of ammonium sulfate to be used in the tissue culture medium depends on the specific plant species and the growth stage of the plant tissue being cultured.
In general, the concentration of ammonium sulfate in tissue culture media ranges from 500 mg/L to 1000 mg/L. However, it is important to note that ammonium sulfate is less soluble than ammonium nitrate, which can make it more difficult to dissolve and prepare the media. It is recommended to adjust the pH of the media after adding the ammonium sulfate to ensure that the pH is appropriate for the specific plant species being cultured.
It is also important to consider the potential toxic effects of ammonium ions on plant tissue. High concentrations of ammonium ions can be toxic to plant cells and tissues, and can inhibit growth and development. Therefore, it is important to optimize the concentration of ammonium sulfate in the tissue culture media to avoid toxicity and to promote healthy growth and development of the plant tissue.
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I have engaged in callus culture since many years, I have interest to know their medicinal properties. If it is, I can use it for other purposes. Sugarcane, plant tissue culture, laboratory practice, callus, nature of callus, Callus induction.
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For that you can create a good suspension culture of sugarcane. If you want to know about the untargeted metabolites you can go for mass spectrometry studies I e. LC MS or GC MS.
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And for PGR solutions?
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At first heat kill the MS media in autoclave and pour it to waste water management pipeline before it get solidify.
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We wish to set up a tissue culture lab but we are constrained by space available to us as a dept (Plant Science & Biotechnology)-We are trying to squeeze in all the lab units into the available laboratory space; tissue culture , molecular biology and plant pathology labs. We have designed the proposed lab with all the units in place taking all the safety precautions into consideration in the design. There is no issue with tissue culture and molecular biology labs all in place under the same roof but my worry is about including plant pathology lab even though it can be accommodated because of the problem of contamination. As the head of department i want to carry everybody along; the plant science ( for plant pathology) and the biotechnology ( for plant tissue culture+molecular biology) staff for logistic reason and otherwise. With a background in biotechnology this is the dilemma I am facing trying to squeeze in all the three lab units against my worry about contamination. Pls i need advise and suggestions from experts in the field of Plant tissue culture , molecular biology etc on this proposed multi-purpose lab. Attached is a sketch of the lab.
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Essien Archibong Okon I worked in commercial plant disease diagnostic lab under the same roof with Plant tissue lab from 2012 till 2021. Finally, we found such location incompatible with stable production of pathogen-free meristemic plants. Do not repeat the same mistake.
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I need examples of asceptic techniques that are used in plant tissue culture.
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The skills needed for plant tissue culture involve learning several lab techniques. I do not know what experience you have, but I suppose you could say that there are 5 phases that you need to know about to be successful. Aseptic techniques are essential to culture plant tissues free of microorganisms.
1. Preparation of solid (agar) or liquid medium. You need suitable nutrients and growth promoting substances and the medium needs to be filtered-sterilized or autoclaved and in sterile plastic or glass containers.
2. You need a clean-air hood running. Wipe the inside with alcohol.
3. Select your plant and in the hood you surface sterilize the plant pieces with a suitable chemical (usually sodium hypochlorite solution). Then wash with autoclaved water in the hood.
4. Cut out inner plant tissues with sterile (autoclaved) dissection instruments or a cork-borer. Use alcohol and a flame to keep the instruments sterile while you are doing this.
5. Inoculate the medium with plant pieces having discarded any parts that were exposed to the chemical used for surface sterilization. Leave in petri dishes or other sterile plastic ware... probably with lights on. In time your plant parts should grow if you used a correct medium.
Good luck.
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During plant tissue culture researcher using citric acid and ascorbic acid to minimise the browning problem of explant. Under which mechanism these two compounds prevents tissue from browning .Please if references provided will be appreciated
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Dark culturing: In principle, light promotes the production of phenolic compounds. Thus, culturing the explants in dark for about 72 hours to a period of 10 days can significantly reduce the chances of browning.
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I am using a specific brand of MS Basal medium with vitamins and I see that my plants are not growing as fast as they should. I am attaching the medium composition of the brand I am using and the one I would like to try. Any recommendations?
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Did not understand if you use the ready MS, one can buy, or you prepare the MS by stock solutions?
Because you pointed to the FeEDTa.
If you prepare this as stock solution together with CuSO4, then you have to cook it! Only then you get the right Fe-solution. You have to wait until the color is changing from light yellow tow dark yellow. Then let cool and fill up.
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we know that Secondary metabolites are non growth associated, and primary metabolites are growth associated, so can the answer be No they have no role? or there are some of them that have role
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Dear Ali Safaa ,
Secondary metabolites are organic compounds that are generally not involved directly in the growth and development of plants but play important roles in their ecological interactions. However, There are some exceptional cases, where secondary metabolites such as alkaloids and flavonoids can directly influence plant growth and development. For example, alkaloids such as nicotine and caffeine can act as growth inhibitors or stimulators, depending on their concentrations and the species of plants. Flavonoids can also play a role in plant growth and development by regulating auxin transport and modulating the activity of plant hormones. Additionally, some secondary metabolites can indirectly affect plant growth and development by protecting them from pests and pathogens, regulating hormone levels, and modulating stress responses.
Nevertheless, it's important to remember that phytohormones, signal molecules, and primary metabolites are the main compounds that directly contribute to the growth and development of plants, while secondary metabolites primarily play roles in plant defense, signaling (here's the overlap), and ecological interactions.
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Hi, I would like to ask what light do you recommend to use for in vitro plant cultures? I use full-spectrum LED light (it is pink shining). Is this appropriate for purpose of regeneration of plant explantats and subsequent micropropagation ? And are there different requirements for light when you try to induce callus formation ?
Thank you for all responses.
Bohuš
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Bohuš Kubala You can get some infromation from Valeria Cavallaro et al.
Light and Plant Growth Regulators on In Vitro Proliferation
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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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Some questions sent by you with the same subject were answered in different dates. So I am in the same posistion as before.
Good look
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Checking of sugarcane production and productivity by tissue culture method, I want to know the answer. As I read and study, this techniques couldn't create genomic modifications. Please, share your views?
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Dear Ravindra Karn yes, it's a fact, and it's named somaclonal variation (see Larkin, P. J., & Scowcroft, W. R. (1981). Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theoretical and applied genetics, 60(4), 197-214.). Certainly, some morphogenic pathways are less prompted to form SV than other; e.g. axillary buds < meristems < adventitious buds < regeneration from calli
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Seeking advice on resolving a problem.
Plant symptoms include:
1. The thickness of the stem and root becomes abnormally large.
2. The thickness and size of the leaves become abnormally large, and the leaves become twisted.
3. Necrosis of new shoots and old leaves (browning or shoot tip necrosis)
Unusually,
in the same medium composition or environment
Some plants grow normally without problems.
I initially suspected hyperhydricity, but
They do not have a glassy appearance.
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Yes, it is a light problem. Seeds germinate in dark or low light conditions, and after this, you need to ensure the roots are not exposed to light and stems and leaves to the correct light intensity and quality :)
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Is there an effective way to detect endogenous contamination (bacteria and fungi) in plant tissue culture? My test method is to inoculate some of the target plants into the Luria-Bertani broth and Potato Dextrose Agar medium to check for bacterial and fungal contamination. However, in some cases, This test method does not work correctly and clearly. Personally, I suspect that there is a limit to the cultivation of various bacteria because Luria-Bertani broth is more optimized for some bacteria. Also, it is inefficient to test twice. Some researchers use a method of testing bacteria and fungal contamination at once using Tryptic Soy Broth medium, is this more efficient and accurate? I ask for the opinions of those who study plant tissue culture.
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Thank you for sharing.
I hope we can share our opinions again next time
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One example: Egg yolks with sodium hypochlorite 20% and tween 20.
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Egg yolks is usually used in tissue culture disinfection because it contains calcium. With sodium Hypochlorite the solution give soft calcium hypochlorite which is a solution against viruses by inhibiting their replication. When Calcium hypochlorite is use for lab material sterilization it is to eliminate the viruses.
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How can we manipulate them without spreading this sticky liquid in our tools and media culture?
As it is the first time I work with milky sap plants, I appreciate all the experienced advices.
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You could try using different source tissues for the micropropagation. Seedlings may have little latex present and would be a good source of fresh growing cells. Seeds are also relatively easy to surface sterilize and grow aseptically.
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I want to understand if there is collaborative relationship between Plant tissue culture and viruses if there is what are them
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Hello!
I am not sure at all that "collaborative" relationship plant-virus exist, but our results proved that the effect of viruses was followed by plant recovery after transgenesis (see reference)
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I am working on micropropagation techniques in wood tree plants, but while doing the endophytic fungi shows dominance, can anyone suggest how to inhibit the endophytic fungi?
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Kumbar Manjunath 1) Follow the recommendations of Ricardo Julian Licea-Moreno
2) Meristem culture helps to eliminate virus, endophytic bacteria, and fungi.
See:
Cassells, A.C. (1991). Problems in tissue culture: culture contamination. In: Debergh, P.C., Zimmerman, R.H. (eds) Micropropagation. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-2075-0_3
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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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Directing Microscope
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I am planning to ask my students to do a plant tissue culture experiment. However, our school does not have a plant tissue culture growth chamber. Does anyone know any alternative?
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Dear Cedrick Alguzar a growth chamber is the place where plant materials growth. Thus, if you select a place to store these materials, this's your growth chamber. But, you could go further with the requeriments. Better to use an isolated place from outer. If you have control of ilumination, it could be fine, but it's not necessary: in Cuba most of tissue culture labs use sun light. Important to have control of temperature. From there on, you can improve your growth chamber, according to your needs and resources
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Hi guys, Is it possible to cultivate interesting mycorrhizal fungi in vitro with in vitro cultivated plants or roots? If somebody has better skills, give advice please. What plants should I use ? Thanks for all responses and advices. Bohus
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As per the INVAM website and literature chicory root will be the best whoever the technology has been tested using roots of many crops like carrot, tomato....
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.
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There are several kinds of gelling agents for tissue cultures like Gellum gum, Gelrite, Phytagel and some Agar-agar (Difco, Merck, and others).
Then you need to choice according the facilities of your laboratory.
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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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Why there are two names for the same growth regulator?
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Yes u can
as chemical constitution of both are same
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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 the 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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Sub culturing multiple shoots induced.
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For tissue culture it is important to consider which kind of plants are you working.
It is very well know that some species have special secondary metabolites that in invitro conditions turn brown. If it is your case, I recommend to use into the media some antioxidants like PVP, Plhuoroglucinol, citric acid, ascorbic acid.
Also you may decrease the light in the growing room.
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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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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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I want to make photographs of my plant tissue cultures for publications.
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The best is to take photos in microscope attached to computer as it has inbuilt software to add scale bars, and also the image clarity is better which is required for tissue culture publications.
But if you don't have that set-up, you can take pictures on any light microscope and simultaneously take pictures of ruler scale at different magnification (which are used for clicking photos).
Then using image J you can add scale bar, it's free software
All the best
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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.