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
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.
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
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.
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
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.
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
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.
Plant tissue culture: ammonium sulfate instead of ammonium nitrate? what concentration? how to prepare stock 1 solution for MS media using ammonium sulfate?
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.
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.
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
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
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?
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.
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?
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)
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.
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.
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.
I want to understand if there is collaborative relationship between Plant tissue culture and viruses if there is what are them
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?
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.
In kang kong, plants are raised both from seeds or cuttings although plants raised from seed is the normal practice. But it has been observed that not all the stem cuttings of kang kong used for propagation exhibited desired performance i.e. produced healthier plants. Only one or two specific nodes exhibited better performance.
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?
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.
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.
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.
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?
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!
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?
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.
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
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?
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…
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.
Prof. Dr. José Carlos Lorenzo Feijoo
Head, Lab for Plant Breeding and Conservation of Genetic Resources,
University of Ciego de Avila, 69450,
Tel. 53 33 225768/212719
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.
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?
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?
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.
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?
Can turmeric be used as a substitute for the other comercially availble antifungal agents during plant tissue culture?