Plant Tissue Culture - Science method

Dear Mr. Ali:

It is not necessary to buy the expensive microscope to do the plant tissue culture in the cell, tissue level. If so, you could match the computer with your microscope similar as for the chip manufacture. The most importance you need to note is of which the valuable plants to do those research.

David

All the best to you with many successes on your research

Dear @Bidisha Mondal You can access the links given below; hope, these could be useful to you.

For laboratory purpose you can can get from any Scientific chemicals company like Quality traders in Hyderabad.

If the cells of dried plant parts (leaves, stem, root, etc) are alive, these can be regenerated.

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:

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)

Dear Mônica Bossardi Coelho

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

Ethanol and water combination. Use filter sterilized.

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!!!!

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.

It is possible from old leaves.

Take a look at this video link

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.

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.

Dear Georgios Formozis

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.

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.

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

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.

Dear Reza Ghahremani

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.

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.

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.

Yes, the nature of the color and its intensity can affect somatic embryogenesis experiments and may result in many differences among the obtained explants.

Robert Samples

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...

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:

Apparently such white spots on Aloe vera can be natural or caused by fungi. I hope this helps. Good luck with your work!

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

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.

Each and every stage and all the observations should be documented as photographs. find a good place for photography, it is an important step as it results depend on or are evidenced by the photos.

Adjust light sources to get good photos. Use a ruler while taking photos and finally you can draw scale bars according to the ruler sizes. If it is microscopic images, scale bars are available in the software itself.

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.

Mahipal Shekhawat of course. A lab in Bulgaria, settled in the Rose Valley (Kazanlak), micropropagate commercially various rose accesions.

Here is the link

Dear José Carlos Lorenzo Feijoo,

Plant scientists at the National Laboratory for Genetic Resources Preservation (USDA), in Fort Collins, Colorado.

Best Regards,

Jean

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

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.

The browning of the culture medium and the tissue is due to the production and oxidation of phenolic compounds from the damaged tissue. To reduce this problem, you can use antioxidant compounds such as citric acid, ascorbic acid, PVP or activated charcoal in culture medium

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.

The attached article showing different steps of an efficient gene transformation method.

Dear Reza Ghahremani culture media with PG just turn yellowish after autoclaving. This colour change seems not caused by sedimentation neither contamination. Other changes can be observed, as softening of gel.

Soaking dishes in hot water with a little soap and sodium hypochlorite has good results. To prevent water stains, we use distilled water for rinsing.

Dear respected Anut,

The images you submited show that you have explant browning in your work. So my first recommendation is to control the production phenol and I suggest adding PVP in medium culture. Due to the tissue browning, it is better to use culture medium 1/2 or 1/4 MS. Also, In my experience, the best hormone to induce callus is TDZ.

If the hormone used is not heat sensitive, the best way is to add it to the culture medium, then adjust the pH and finally autoclave. If the hormone is heat sensitive, you can test the rate of change and adjust the pH accordingly before autoclaving.

Optimal protocol in woody plants means the best performance at the tissue culture stage (establishment, multiplication, rooting). As we know, most of the growth parameters in these three phases do not need to destroy explants and plantlet, such as the number and length of roots and stems, the time of the first emergence of adventious roots, survival rate, responding explant percentage and etc.

Dear respected Kamila Kozak-Stankiewicz,

From what you have said, it is understood that the in vitro and acclimatized plants were are contaminated, and this can be an indication of endogenous contamination. On the other hand, the use of alcohol alone can not have a complete effect on field mother-explants. I suggest you to use sodium hypochlorite (20% ) supplements with tween 20 as a surfactant and then washout three times with sterile distilled-water (3,5,2 min, respectively).

Certainly, each method, despite its advantages, has its challenges. The history of using this method has shown that its benefits far outweigh its problems, and of course most of these problems have been solved. All the problems mentioned above are absolutely true, but the advantage of this method can not be ignored. The plant tissue culture method has been very helpful in preserving endangered plant germplasm as well as their genetic trasformation and plant physio-biochemical mechanisms identification. Other benefits of this method include: A) Callus induction; B) Embryo formation and proliferation; C) Embryo maturation; and D) Embryo germination E) Massive clonal propagation of genotypes of interest F) Production and propagation of disease-free plants by tissue culture G) Embryo rescue H) Management and modification of the ploidy levels I) Germplasm conservation J) secondery metaboilites production and etc.

If the volume of soil used is not large, an autoclave or micro-oven can be used for sterilization.

I have not had the experience of using turmeric as an antimicrobial agent in PTC. In our country, this substance is much more expensive than conventional disinfectants. On the other hand, we know that turmeric has antimicrobial properties, but it is a natural phenolic compound and its negative effect on cell growth and proliferation has been reported. Also, in my opinion, since this yellow substance reduces the transparency of the culture medium and the environment becomes opaque, I preffer to use the transparent materials.

I recommend Invitrosoft (IVS) software because you can use it in a tissue culture research with many option.

In my experience, Silver nanoparticles is successful at 200 ppm concentration for 10 minutes.

In my experience, Phytagel and Agar is more effective than others.

As far as I know, 45 μmol m–2s–1 light is required for plant growth under in vitro circumstance.

Dear Swati,

Please take a look at these 2 interesting papers attached. I hope it will be useful for the discussion.

Polyethylene glycol and abscisic acid improve maturation and regeneration of Panax ginseng somatic embryos

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

Best regards,

Jean

Actually, the OD measurement record of Agrobacterium depends on the type of tissue or explant and method employed, but mostly OD600 from 0.2-0.6 can be used.

Dimethyl sulfoxide (DMSO) as an important polar aprotic solvent that used to resolve sensitive substance that reduce their capability under autoclave heating. Because DMSO is toxic to microorganisms, if it is added will sterilize the culture medium (after autoclaves).

Do you need it for experiment or just for growing plants?

Actually, for acclimatization, plants don't really require very accurate humidity and temperature control. You only need an accurate one for experiment. It will also depend on your facility, e.g. how big your room is, whether air from outside can get in and affect the control of temperature and humidity, presence of cold area that could cause water condense, tolerance of other electrical apparatus to mist, etc. But in general, for tissue culture you can go with any type you can find. In fact, I would say buying separate thermostat and humidistat and connect them to normal heater and humidifier respectively would give you better control than buying a smart humidifier.

Dear @Gaurav Mudgal

Tissue culture protocols may vary depending on plant species. You have not mentioned it. Why are you interested in "unusual" method? Are you working on variant method? If so, you may standardize it. If you are interested in references, use correct key words, and google; you will find so many articles and references.

Please have a look at these PDFs, probably you could find them useful.

Just be sure to use sterile water for any rinses, no point in doing all the hard work to try and get surface sterile plants just to add back contaminants from non-sterile water.

Dear @Badhepuri Mahesh Kumar Contaminants in tissue culture laboratory are most commonly of biological nature, and can include bacteria, fungi, viruses and parasites. For each category, a separate disinfectant is required. For example, the work surface should be decontaminated with antifungal agent (5% Trigene) followed by 70-80% ethanol as antibacterial agents.

Although ethanol solution (70 or 75 %) is used widely as a disinfectant in cell culture room, the experiment or should be careful on the quantity sprayed and where to spray it, and particularly to avoid its contact with experimental cells, since this will lead to radical influence on cells pathophysiological condition.

Please also check a similar discussion at RG; hope, it may throw important insight for this discussion too:

Best wishes, AKC

The response of the cells present in different parts of a plant may vary in vitro. Because of the variable availability of PGRs in different organs, the explants respond differently to exogenously supplied PGRs under in vitro conditions.

We are using Plant Gel at the place of agar, it is a cost-effective and transparent gel and a very good alternative to the agar.

Dear Abdul Main Although plant tissue and cell culture is performed in aseptic conditions, sometimes contamination occurs. Please go through the links given below; hope these could be helpful to you:

In addition, a similar question was put up earlier at RG; hope, answers to that related question might also be helpful to you:

Best wishes, AKC

Water temperature is very important I agree with Mohiuddin Yatoo

Dear @Amina Ilyas You may find a detailed discussion related to your query at the link given below as to why 70% ethyl alcohol is used for surface sterilization:

I would like to add that I am fully convinced with the explanation by @Yuan-Yeu Yau on that thread.

Tween 80. Maybe Triton-X-100. Especially Tween 80

از اطلاعات کلیهدوستان سپاسگزارم

Settanan Plangsiri

1. I am not sure I completely understood what you meant by "it's not a 100% yield right?"

2. The 3 statements you listed are all possible

3. <by putting in the antibiotic, you were able to select for only transformed cells.>

That is a textbook standard theory (the textbook would tell you so). But, In reality, you might get *chimeric* cell clumps, meaning a clump of cells constitute both transgenic and non-transgenic cells. This is might due to 'protection effect'. For some reasons, the transgenic cells can protect the neighboring non-transgenic cells from dying due to antibiotic selection.

4. Several things you don't want from your generated transgenic plant lines: (1) chimeric (not a homogeneous transgenic plant), (2) multiple copies of transgene (which might lead to gene silencing), and (3) sterile, such as no seeds (no progeny), (4) transgene can not transmitted into next generation

Yuan-Yeu Yau Take a look at the paper by O. Rosspopoff et al. (2017) that I attached in my reply to Settanan. (DOI: 10.1242/dev.142570)

Another important work is by Kareem et al. 2015 (10.1016/j.cub.2015.02.022)

And you might want to take a look at one on these review papers:

Radhakrishnan et al. 2018

Sugimoto et al. 2019

and Ikeuchi et al. 2019

I have used Ethanol in dilution only. Spirits are dehydrating and can dessicate the plants. Usually bleach and Tween 20 are used for my plant samples. Be sure to add a sufficient amount of water for washes between washing with ethanol.

As mentioned already by the RG members following this thread, pH influences the availability and uptake of nutrients and plant roots' growth.

Dear Titus,

Please take a look to this manuscript https://doi.org/10.1155/2020/8853426

(It is open access). The authors did quantitative analysis of quercetin in different samples using an HPLC system

I have attached other papers on qualitative study of quercetin in in vitro plants.

Best regards,

Jean

ppm

Dear Kara

These two articles could be beneficial.

Warms

Thanks

Thank you Dr. M. Çağrı Oğuz and Dr. M.K. Tripathi for your attention

By propagating ornamental plants

Good day. To prepare the culture medium you need to start by deciding on the concentration of your stock solutions for your chosen medium. Most researchers usually prepare four components (stocks) of the culture medium: which are macro elements, micro elements, vitamins and chelate (iron stock). In most cases preferred strengths of the highly concentrated stocks are 20x for macro elements, and 200x for macro elements, vitamin stock and a chelate stock. The dilutions are made from these concentrated stocks as follows, for a 1litre culture medium: take 50ml of the 20x macro element stock and 5ml from the other 200x stocks (micro, vitamin and chelate). This is basically how the media are made. The other components are made and added after having made these stock dilutions. These components are usually hormones, sugar sources like maltose, sucrose etc, solidifying agents like gelrite or phytagel, casein hydrolysate, and so forth. Some hormones are heat labile and are added after autoclaving the medium, one needs to know how stable are the hormones when exposed to high temperatures. The pH of the medium is adjusted using 1M NaOH or HCl before autoclaving. The medium is autoclaved at 121 degrees celcius for 20 mins, and after cooling down an amino acid L-glutamine is added before dispensing the medium in a sterile laminar flow bench. My humble apologies that I do not have a reference material as per your request however feel this summary will help you make your medium without much difficulty. This is a detailed plant tissue culture medium preparation procedure.