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.
Questions related to Plant Tissue Culture
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.
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?
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.
Low cost easily available gelling agent
From where we can procure Ammonium Nitrate (NH₄NO₃) for Plant Tissue Culture medium preparation?
Please suggest some leads
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.
Various plant growth regulators were found to have a positive effect on organogenesis and somatic embryogenesis, but which one is more important?
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 !!
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.
How to prepare Dicamba stock solution for initiation of callus in plant tissue culture?
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?
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.
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?
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.
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.
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?
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?
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?
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?
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!
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.
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
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
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?
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.
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.
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.
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?
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?
I want to make photographs of my plant tissue cultures for publications.
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.
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?
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
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...
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.
Sub culturing multiple shoots induced.
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 ?
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.
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.
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
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.
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.
Non-destructive ways to measure (quantify) micropropagation "success" in woody plant tissue culture
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).
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?
agriculture, soil, mycorrhizal fungi, biology
Can turmeric be used as a substitute for the other comercially availble antifungal agents during plant tissue culture?
A software which can help to design the experiment, store data, update new data, set subculture reminders, barcoding etc. Like Invitrosoft.
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?
Intensity of light required for plant tissue culture?
What are the roles of Mannitol, Polyethylene glycol ( which one 4000/6000/8000) and Absicisic acid in somatic embryogenesis in callus for plant regeneration?
How can DMSO be used to obtain disease free material in plant tissue culture and sterilization of plant growth regulators?
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.
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 .
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!
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.
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?
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.
Is anyone used hydrogels instead of agar or gellangum as matrix for micropropgation/plant tissue culture experiments
Especially in Growth Chamber, we found same days culture some are good and some are attacked, Why ? and what are the Causes?
How can I wash it? Please suggest how.
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?
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?
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.
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)?
I am wondering whether it is possible for two callus to be combined into one... both between genetically similar plants and genetically different plants.
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
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?
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
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.
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?
Could it be starting of callus tissue? Why it look white color?
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?
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.