Science method

Micropropagation - Science method

Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.
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I have recently decided to start a large-scale Vanilla planifolia micropropagation and need some suggestions around medium options, environmental conditions, phytohormone supplementation, and other important points on shoot proliferation, rooting, and acclimatization stages.
Thanks for sharing your experiences.
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Yanina Aldao Galván Best wishes, I look forward to knowing your valuable experiences.
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Dear fellows
I am into micropropagation of calladium. we didn't observe any bacterial contamination during the initiation and first subculture stages. the second subculture was done last week and found that all my cultures are bacterial contamination after one week.
the media contained 2% PPM also. is there any advice so I can save my explants?
or all should be autoclaved?
I am really devastated by this bacterial contamination we frequently face.
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It is almost impossible to rid cultures of contaminants. The best approaches are to start by having very vigorous mother plants and a clean growing environment. Only take explants from rapidly growing plants. You might try to treat the explants with ppm before placing them in culture. See Kushnarenko et al 2022. Then monitor the new explants on nutrient agar (see Reed et al publications). By selecting clean explants early in initiation, you can sometimes get a few clean propagules from dirty mother plants and multiply from them.
For your contaminated explants, try removing them from culture and shaking them in ppm solution, then replant on medium. It might not work, but will at least slow down the contamination.
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hello everyone
hope everything goes well in your life.
has anyone had a experience in micropropagation of caladium?
I have recently decided to start a large scale caladium production unit and need advices.
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Rapid propagation of Caladium bicolor was achieved in vitro. Excellent results for shoot induction from apical meristem were obtained when it was cultured on MS medium containing 1mg L-1 BAP within 8 days of inoculation. For shoot multiplication maximum number of shoots was obtained when 0.25 mg L-1 NAA was added to shoot induction medium i.e., MS medium supplemented with 1 mg L-1 BAP + 0.25 mg L-1 NAA. Cent percent rooting was achieved by transferring an individual microshoot to modified MS medium containing combination of 2 mg L-1 IBA + 1 mg L-1 NAA. Plantlets were successfully transferred to greenhouse in sterilized sand and nourished with Hoagland's solution. After acclimatization these plants were shifted to natural conditions in pots containing growth mixture (sand + clay + peat at 1:1:1 ratio), where 100% survival was observed. We concluded that technique of micropropagation can successfully be used for large-scale production of premium quality planting material and almost 2500 plants of Caladium could be generated from single apical meristem in a 12 weeks period.
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needed to find resources about using best medium (MS or MT) for Citrus micropropagation
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Murashige and Tucker Medium (MT) is a modification
of Murashige and skoog medium which contains
increased level of vitamins like thiamine, pyridoxine,
nicotinic acid and sucrose responsible for favorable in vitro
growth of citrus species
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Good day,
i have a general question about tissue culture.
I have found the following recipe for Epipremnum Aureum "Marble Queen":
Leaf Explant: MS Medium + 4.54 µM TDZ + 1.07 µM NAA (Thidiazuron in Micropropagation of Aroid Plants by Chen and Wei (2018), p. 105, DOI: 10.1007/978-981-10-8004-3_4)
Specifically, I have the following questions.
1) Do i only need to autoclave the agar with distilled water (I use a pressure cooker for this) and when the agar has cooled down a bit just add the MS, TDZ and NAA and mix it or do i need to autoclave the MS as well?
2) Will the TDZ dissolve in the agar water at all and how hot can the agar water be to add the MS, TDZ and NAA?
3) Is it even necessary to autoclave the water incl. agar (in the pressure cooker) if I clean all the jars with NaClO (sodium hypochlorite)?
Thank you in advance!
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You could made the MS medium and pour in to the glass jar and autoclave with your pressure cooker. Once it cooled down add the hormone by filter sterilization. Good luck
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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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We often face the problem of fungal Contamination of perrenial crops, tree crops. We used different pricedures for pretreatments and surface sterilization. However, the endophyte fungi is still a Major problem for Tissue Culture of tree crops. Could you please share your positive experience how to overcome heavy endophyte infection to establish reliable micropropagation protocol for tree crops?
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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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Dear all,
Since Larking and Scowcroft coined the term somaclonal variation (SV) has passed a long time. Regardless it's considered a variation source, with a potential great repercussion for creation of new plant varieties, to the best of my knowledge, there's not registered varieties from SV. As likely I'm not so well informed, I would appreciate some information about it.
Thanks beforehand
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Somaclonal variation is seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. ... Therefore, it can be defined as the variation that occurs because of genetic mutation caused by in vitro conditions or by chimeral separation. https://www.sciencedirect.com/topics/immunology-and-microbiology/somaclonal-variation
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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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Hello everybody,
There is a problem with Ziziphus jujuba micropropagation through the single node and shoot tip explant culture. The explants suffer from leave shedding, shoot tip necrosis, and dieback within two weeks of culturing on multiplication media. Until now, different multiplication media, including interactions of different nutrient media (MS, WPM, WPM, and QL) with different PGRs (BAP, Kin, TDZ, NAA, IBA in different concentrations) along with various complementary materials (Fe-EDDHA, glutamine, Ca-gluconate) were tested and, unfortunately, they have not been effective. In addition, we tested the media with manipulated CaCl2, Ca (NO3)2, and H3Bo3 (up to doubled concentrations). However, none of the treatments have been effective so far.
You can find some photos of the explants in the attachment.
I would be grateful if you share your experiences in this subject.
Best regards
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We also observed similar problems in certain Lamiaceae members and got rid of the problem using meta Topolin and doubling the calcium and boron concentrations. I would suggest you use meta Topolin and a slight increase in calcium and boron concentration in the medium can reduce the shoot tip necrosis.
Please go through the following article for reference,
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Hi, I'm going to make a micropropagation and I want to add to my MS Media 2mg/L of activated charcoal as an antioxidant, but I want to know the best way to prepare the solution, I know that it's insoluble in water.
Thanks
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Hello Julia,
thank for posting this interesting technical question on RG. Activated charcoal is a form of (impure) carbon with a high surface area. To the best of my knowledge, activated charcoal is not only insoluble in water but also in all organic solvents. Thus (unfortunately) the answer to your question is that you cannot prepare an activated charcoal solution. You can either use it in suspension of add it as finely powdered solid in the desired amount.
P.S. It might be possible to "dissolve" activated charcoal in so-called piranha solution (i.e. a mixture of concentrated sulfuric acid and hydrogen peroxide). However, once it is dissolved, it isn't activated charcoal any more....
Good luck with your work and best wishes!
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Hello everyone,
I'm working on micropropagation of Juniperus spp. I have faced with a big problem. Two or three weeks after establishment of cultures the bottom parts of explants turn brown which leads to growth inhibition.I'm looking for the probable reasons and suitable solution.
Please see the attached photo.
I would be grateful if you could kindly help me.
Regards
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Ruhollah Abdolinejad, all the tree sp. have this problem generally and this is due to phenolic leaching from the explants. Treat the explants with antioxidant solutions before inoculation and the addition of some antioxidants in the medium will solve the issue. For details read some of our articles on the tissue culture of trees you will get an idea about it. All the best..
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Dear Colleagues,
I would like to ask the following question. In an experiment to reduce hyperhydricity in TIS bioreactors for plant tissue culture, I added 100 and 200 mg/l Phloroglucinol (Duchefa Biochemistry) to liquid medium. Unlike other (colourless) media, the medium with phloroglucinol always colored bright yellow after autoclaving. Although Phloroglucinol is described in the literature as autoclavable (e.g. Teixeira da Silva et al., 2013: "the anhydrous form melts only at 218-220 °C"), I wonder to what extent the degradation (resulting in a color change) of PG affects the efficacy of this compound?
Curious to hear about your experiences.
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Dear Reza Ghahremani culture media with PG just turn yellowish after autoclaving. This colour change seems not caused by sedimentation neither contamination. Other changes can be observed, as softening of gel.
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I'm trying to root in-vitro grown walnut, but the rooting percentage is extremely low. The plants were multiplied using DKW salts (Driver and Kuniyuki,1984) supplemented with 30g/L of sucrose, 4.4uM 6-benzyladenine and 0.05 uM IBA for 2 weeks under 16-h photo period. Then, i put the plants on root induction medium using DKW salts (Driver and Kuniyuki, 1984) supplemented with 40g/L of sucrose and 50uM K-IBA in the dark for 5 days. Following the root induction, shoots were put into fog chamber to root ex-vitro.
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Dear respected Anut,
The images you submited show that you have explant browning in your work. So my first recommendation is to control the production phenol and I suggest adding PVP in medium culture. Due to the tissue browning, it is better to use culture medium 1/2 or 1/4 MS. Also, In my experience, the best hormone to induce callus is TDZ.
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Non-destructive ways to measure (quantify) micropropagation "success" in woody plant tissue culture
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Optimal protocol in woody plants means the best performance at the tissue culture stage (establishment, multiplication, rooting). As we know, most of the growth parameters in these three phases do not need to destroy explants and plantlet, such as the number and length of roots and stems, the time of the first emergence of adventious roots, survival rate, responding explant percentage and etc.
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Can turmeric be used as a substitute for the other comercially availble antifungal agents during plant tissue culture?
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I have not had the experience of using turmeric as an antimicrobial agent in PTC. In our country, this substance is much more expensive than conventional disinfectants. On the other hand, we know that turmeric has antimicrobial properties, but it is a natural phenolic compound and its negative effect on cell growth and proliferation has been reported. Also, in my opinion, since this yellow substance reduces the transparency of the culture medium and the environment becomes opaque, I preffer to use the transparent materials.
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Hi dear fellows
I am working on micropropagation of Begonia and Gloxinia which in both of them have internal bacterial contamination.
I tried ppm and antibiotics in media but both of them have negative effect on multiplication and growth of explants. As I am a mass producer of these two plants this contamination costs me a lot every year. do you have an idea that can help in controlling and eliminating endogenous bacterial contaminatin?
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In my experience, Silver nanoparticles is successful at 200 ppm concentration for 10 minutes.
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Hi Anusha, hope you are doing great and staying safe. Looking at the bamboo images above (Test Method 3), I am really impressed and curious. I am a Ugandan working on a similar project, I am tasked with developing protocols for micropropagation of 3 bamboo varieties (Dendrocalamus giganteus (Giant Bamboo), Oxytenanthera abyssinica (African low land bamboo) and Bambusa vulgaris (Golden/yellow/common). I have however encountered a number of challenges but majorly contamination (fungal and bacterial) of initiated explants and browning of successfully regenerated bamboo shoots (Picture attached). Please advise how I can solve these problems, thanks a lot in advance. Tuhaise Samuel
PS;
I am currently using this media composition for initiation and micropropagation [MS + BAP (5mg/L) + 2, 4 D (1 mg/L) + Vitamin C (5mg/L) + TDZ (0.5mg/L)]
Do you use antibiotic in media?
How do you maintain your mother gardens (do you spray with a bactericide and how regularly?)
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You can reduce phenol content in addition to subculturing by adding Polyvinylpyrrolidone (PVP) or ascorbic acid in medium culture. Of course, images clearly show that the leaves of the plants became yellow, and this can be attributed to the inability to control phenol and inappropriate plant medium. So I suggest using several culture media or reducing macro salts, make the optimize conditions for this plant.
Finally, I recommend the study of the attached article.
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I am working on propagation of Stephanotis floribunda , Madagascar Jasmine, I don't find a reference for information about micropropagation of this species . Picture is from my culture
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Thank you
Dear Kourosh Vahdati ,I use MS medium with Ms Vitamins
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As you know, Monstera is very popular house plant and some has unusual mutation with reduced number of chlorophyll and in some cases, chlorophyll pigments completely lost in leaves. Is it possible to make chlorophyll degradation in leaves under lab conditions?
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The use of antibiotics, preservatives and similar chemicals is aimed to be avoided in cultures. Since isolations without the use of antibiotics and preventive chemicals is my goal, latent bacterial and sometimes fungal contamination is encountered. Does anybody has experience treating source plants with any preventive chemicals or antibiotics few days or weeks prior to isolation to reduce or eliminate the internal contaminants?
Any recommendations/suggestions will be highly appreciated.
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Just be sure to use sterile water for any rinses, no point in doing all the hard work to try and get surface sterile plants just to add back contaminants from non-sterile water.
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On the same medium composition i.e. 2mg/l 2,4-D and 0.4/0.5mg/l Kn MS medium(1962). It is found that leaf and petal explant gives embryogenic callus and node, internode and petiole derived explants with nonembryogenic callus.
In the same study I have performed one more experiment: node, internode and petiole derived callus were transferred to cytokinin rich medium for shoot regeneration; after one month shoot bud differentiation was observed in all the calluses (node, internode, and petoile derived callus) but there is significant difference in morphogenetic potentials of each explant derived callus. I have further subcultured each callus maintained separately and studied for 7 subcultures... during subculture on callus maintenance medium I tried the same composition. During each phase of subculture I have studied morphogenic potential of node, internode and petiole derived callus.. during this study I have found that petiole derived callus retain morphogenic potential up to six subcltures, while the other explants fail after 3rd and 4th subcluture... I need references for a similar study. If anybody has any suggestion please recommend some research papers on related work.
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The response of the cells present in different parts of a plant may vary in vitro. Because of the variable availability of PGRs in different organs, the explants respond differently to exogenously supplied PGRs under in vitro conditions.
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How can I wash it? Please suggest how.
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Water temperature is very important I agree with Mohiuddin Yatoo
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Although it's not a very effective sterilizer alone for plants but still being part of treatments in all researches. i wonder over its widely essential use and what factors decide the time of ethanol treatment?
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Dear @Amina Ilyas You may find a detailed discussion related to your query at the link given below as to why 70% ethyl alcohol is used for surface sterilization:
I would like to add that I am fully convinced with the explanation by @Yuan-Yeu Yau on that thread.
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I am working on the micropropagation of Tea, I need to speed up the growth of my seedlings in a short period of time. I need to know which kind of hormones I should apply in my growth medium. I was very thankful you are considering my problem.
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Many hormones required for cell growth, such as auxins, gibberellins, brassinosteroids, ethylene, jasmonates, salicylic acid, strigolactones and cytokinins which able to accelerate or promote growth...
Auxins
Auxins promote stem elongation, inhibit growth of lateral buds (maintains apical dominance). They are produced in the stem, buds, and root tips. Example: Indole Acetic Acid (IA). Auxin is a plant hormone produced in the stem tip that promotes cell elongation.
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Hello.
We optimized a protocol and media composition for tissue culture of Abaca (Musa textilis Nee) using the varieties we have on the facility. However, upon using different varieties from the collection obtained all over the country, the explant seems to be unresponsive. No shoots formed and severe browning (may be caused by phenolic compounds) could be observed.
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Answer was send on the las 23 March with several recommendadionts according the Musa spp. genome .
You will see it is working, successes.
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i would want to also know the uses of such chemicals and regents
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pH Meter
Laminar Air Flow
Autoclave
Freezer
A scale with two decimal places and measures in grams
Containers
Beakers
Flasks
Volumetrics
Pipettes
Graduated cylinders
Gelling agents
Medias
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Hello everyone! I am looking for some statistical help!
I am working in in vitro micropropagation and one of the aspects that I am studying is the rooting of plants in 10 different growing media. For that, I categorize the rooting of the plants as 0 (no roots) or 1 (rooting). Therefore, I have one categorical independent variable (rooting medium) with 10 different categories and one categorical dependent variable with two categories (0 and 1). My sample size is 30 plants per medium.
From those numbers, I obtain a rooting percentage for each medium and I would like to now if this parameter differs significantly between the different media.
I would like to know how I should compare my data. These are the tests I have done but do not know which one to use:
- 2x10 contingency table in SPSS and Chi-2 test. This gives me a p value for the whole experiment. For post hoc, I have used a Z-test.
- I have also tried a univariate GLM in SPSS with a tukey post hoc test.
I do not know if both are valid or if I should just stick to the contingency table.
Any help would be appreciated
Thanks!
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Maybe I am reading this differently from Jochen Wilhelm and others, so am taking a different tact (but am certainly willing to be convinced that I am wrong ... and this is not my substantive area, so ... ). My concern is with a statistical approach that has 9 values that are the focus and the power of post hoc tests with this many categories. In order to offer guidance, I have a question.
Do you have any ideas/theories/hunches about the relationships among the 10 categories?
If yes, use these ideas in you model to focus what you are looking at.
If not (so these are just random categories that were picked), presumably your interest is just whether there is more variability among these categories than expected if the categories don't make a difference. Then, you'd probably want to allow the intercept to vary by category, and might assume the distribution of these intercepts is random, and then just have a single number (the variance) that you are focused on.
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One of the main issues in better acclimatization of in-vitro woody plants is "stem thickness". How can we get the ticker stems during multiplication stage?
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Not only the light intensity but also the the quality/source of light influence the growing shoot..for example:BLUE light strongly inhibited the elongation. Incandescent light promoted both growth of stem diameter and elongation of leaf petioles (From:Reference+my own experience)
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We are going to start micropropagating cannabis on 2021, and we are thinking in using some kind of biorreactor to scale the production, does anybody is working on this theme? Can you share info with me?
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I don't think bioreactor system would be efficient for Cannabis micropropagation. First of all, you have to optimize it solid culture, and try to minimize gelrite/Agar concentration whether your genotype is fine for it or not. Then you can try temporary immersion system with suitable genotypes. But of course, there are a lot of risks in each set up, you have to calculate the costs for sustainable production to decide the way of scaling up.
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Cannabis micropropagation
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Thanks for answering. I still seek someone who has a protocol that is repeatable with cannabis, which is uniquely incalcitrant. The numbers of plantlets produced remains unsatisfactory for high frequency regeneration from callus, despite numerous combinations of plant growth regulators.
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Dear peers,
I am currently making a research proposal on conservation of some endangered Ceropegia and Brachystelma species. Please suggest some advanced models/methods/papers to evaluate the soil, water and climatic requirements for both micropropagated and naturally grown endangered plants.
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studying the agro-pdeological conditions of naturally grown endangered planst and later underatking in-vitro multiplication is quite task . These two exercises have heaven to hell difference. But , all the best to you....
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I am doing a study using cotyledons as the explant to mass propagate forest tree plants through micropropagation. I experienced only rooting in all the treatment with auxin and cytokinin (BAP, IBA). I need to initiate shoots. Is there any alternative method for shoot initiation through cotyledons?
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I agree, cotyledons are a known source of auxin which is then transported to the petiole. That could explain your difficulty to regenerate shoots. You should take that in consideration and adjust your phytohormone ratio in favor of cytokinins.
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Can i use intercalary meristem on the bamboo shoot as explant for indirect somatic embryogenesis?
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You can do it. Please see the following articles. I wish you good luck.
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Analyzing different concentrations and combinations of growth hormones on axillary bud proliferation, elongation and rooting
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Yes, the main factor may be growth regulator as example and explant type, media type, sugar concentration or medium concentration as secondary factor.
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Cultured on MS media with TDZ. TDZ @ 0.5 to 7.0, but lower concentration of TDZ i.e. 1 to 3 mg/lt is showing yellowness of leaf explants
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i think that the shoots is direct organ-geneses
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For Rosa damascene micropropagation, Use of Which Growth Regulators is to Increase the Number of Shoots Extracted from the Stem Node?
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Hello
Appropriate growth regulators treatment depends on some factors such as accession, growth conditions and mother plant age. In the articles, combination of 2.5–3 mg/l BA + 0.1 mg/l IBA, 3 mg/l BA + 0.1 mg/l IAA, 5 mg/l BA + 0.1 mg/l TDZ, 2 mg/l BA + 2 mg/l GA3 and 1-2 mg/l BA + 0.1 mg/l GA3 ± 0.1 mg/l NAA were the most suitable treatment for proliferation.
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I'm doing a research about tissue culture and I think I don't have enough knowledge about it. What are the problems that I will face on my research and what are the solutions? What part of the plant will I use? Will I use MS medium or other mediums? What disinfectant will I use? I hope you can help me and thank you in advance.
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Dear Gerrard
You can use the micropropagation protocol of other species like Tectona grandis.
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I'm planning to research for finding a micropropagation technique for an endangered tree. I'm worried because we are only given limited time to conduct the research. Thank you for your time and response.
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Three months really isn't enough time to do the kind of project you are talking about. At best, you might be able to get through 1 or 2 micropropagation cycles, and that is assuming that the shoots respond well to the media and you don't run into any obstacles (such as contamination or browning). I'm not very familiar with the Tectona genus, but unless the shoots grow rapidly in culture, it could take several subculturing cycles over many months to get reliable results. It might be possible to get through a simple experiment, but any complex project would most likely take more time than you have available. It's up to you if you want to take that risk or not.
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When determining subculturing time, what markers such as whether the explant shoots are green, callus size, etc.?
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We are working to propagate some citrus rootstock in vitro. We noticed that subculturing every 4 weeks is better in growing plants because the medium of cultivation during this period is depleted. However, the period of subculture depeded on the plant species and culture conditions.
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I am doing papaya micropropagation right now with apical shoot as plant material, but after two weeks of initial culture, the growth of explant is hindered by softening tissue (pale in color) on the middle portion of explants.
Can anyone advise on how to avoid softening tissue in papaya micropropagation ?
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Dear Researcher
In response to your question, I am to suggest you take MS medium along with 1.5mg/l BAP and 1.0 mg/l gibberellic acid for initiating culture. Second point is that the explant/seeds not to expose high concentration of sterilant solution.
Best wishes
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What are the effective materials for its capacity on the absorption of phenolic compounds excreted from explant cut, to prevent the occurrence of browning during tissue culture and micropropagation experiments?
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@ S. Wenkart .. hey, Dear, I worked most about aromatic and phenolic compounds and biodegradations, but in new my research I was looking an effective method for prevention of occurrence of browning and reduce the effects of phenolics during micropropagations for obtaining higher efficiency...
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Is it possible to use PPM (TM) with antibiotics to eliminate endogenous bacterial contamination ?
PPM Alone or even Antibiotics alone can't really give a good percentage of endogenous contamination free explants for mass propagation . i am interested to know if PPM and antibiotics can be used in combination to increase efficacy of this treatment.
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Sometimes the problem is not the substance, but the method that is used for treatment. I'm not expert in endophitics; however, I'm dealing with it every day, as every tissue culturist. I see quite difficult to obtain 100% bacteria free plants. If your objetive is to micropropagate, my advise is to focus your attention in to control the contamination, not to produce 100% bacteria (there're also fungi....) free plants. Microorganisms have evolveld together with plants, so both need each other in a symbiotic relationship; therefore don't underestimate it.
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Need of experts suggestions regarding somatic embryogenesis in this plant family. Kindly post your valuable opinions...
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Plant tissue culture system are effectively used in commercial production. Of those, African violet is one of the best example for cost effective production. After emergence of new tecnologies such as LED systems in Plant factories, selection Of best cultivars for certain plant species urges us to find cost effective solutions. Could you please share any ornaments/decorative species that are available for the simplest way of production through a tissue culture system?
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Anthurium andreanum
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I am working on Papaya micropropagation where I am getting plenty of shoots but they are not increasing in height. I have tried diffuse light, GA3 but did not see improvement. Please suggest a better treatment.
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Dear Parth Desai .....
you can decrease the Light intensity for a week in first sub culture, or also use low concentration of GA3, that will effective in elongation of your new shoots
Best regards
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What are the durabilities of common plant growth regulators in solution when stored at a proper temperature and dark?
Especially for GA3, IAA, NAA, BAP/BA, BPA, kinetin, adenin
Many thanks in advance
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Good question. Some hormones are more chemically stable than others. But always storage in a fridge up to 6 C. For instance, 2,4-D is highly stable and lasts for months, IAA very liable, Zeatin, worst. Zeatin must be stored at -20 C and used immediately. I my lab we use to renew from two weeks to a month for most of them.... Again, keep eyes on the solution before using it and make sure your fridge is always working properly. One weekend without electricity may damage your stock solutions...
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I need to use this technique to micropropagate a great number of apples
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Dear Sabry,
You can try to use 0.1 μM TDZ for triggering axillary shoot proliferation from apical or nodal explants. TDZ is eficcient PGR for woody plant tissue culture.
And I adree with Dr. Ricardo Julian Licea-Moreno, there are the published papers on this topic, for instance
With best wishes,
Yulianna.
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Hi,
I was wondering what is the best way to preserve IBA, TDZ and NAA once in solution.
Thank you
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Ideally at -20 ° C and in small aliquots (1mL) in Eppendorf tubes.
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Dear Research gate community,
I was wondering if any of you add a pH indicator and/or antioxidant to the culture agar media to produce explants.
Thank you
Nico
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Of course, important changes in pH of culture media might occur during the culture. I don't have references about indicators for carbon source depletion, although, great changes also occurr in mixotrophic cultures.
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Is the temporary immersion system used in the production of zamifulia? What kind of system is useful for micropropagation ?
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Hello, yes you can find the answer of question in attached paper.
Good luck!
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Methods of micropropagation are broadly divided into two categories— 1. Axillary regeneration (Shoot tip and node culture), 2. Adventitious regeneration (somatic embryogenesis, and organogenesis).
Again the second category i.e. adventitious regeneration (both embryogenesis and organogenesis) may go through either direct morphogenic path or through indirect morphogenic path via callus phase.
If their is any other in vitro method of micropropagation are available, which are not fall in any of the above mentioned categories, then please discus.
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In micropropagation, we could produce in vitro plants by:
(1) meristem, shoot tip or nodal culture
(2) plant organ (leaf blade, petiole, rhizome, root segment, etc.) culture
Both (1) and (2) can give us the whole plant, directly or indirectly, depending on the culture conditions (chemical or physical).
Somatic embryos can also be induced, directly or indirectly, by these culture methods .
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I work at Center for Advanced research in Plant Tissue Culture, Anand Agricultural University, Anand, India. Currently I am woking on micropropagation of Sandalwood. I have successfully completed multiplication but finding difficulties in rooting phase. I have experimented various approaches for in vitro rooting such as higher auxin levels, combinations of auxins, pulse treatments, various hosts, biotic as well as abiotic stress but none of them have proved viable. The frequency of getting roots is very low (only 1 at every 100). Please suggest a reliable method for high frequency rooting.
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Tripathi et al High frequency in vitro regeneration of sandalwood (Santalum album Linn. September 2017. Medicinal Plants - International Journal of Phytomedicines and Related Industries 9(3):154
please refer to the above
also, as against the previous beliefs it is been reported that CK has an equally imprtant role in rooting induction as auxin, please refer to one article "releasing cytokinin brakes on rooting" which featured in journal of plant physiology, this issue or the previous one
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Do anyone know about micropropagation(tissue culture) method of guzmania plant?
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In a study, Guzmania lingulata protocorms were used as materials to establish the protocorm culturing system of Guzmania lingulata. Adventitious shoots could be efficiently induced from axillary bud,terminal bud or leaves in the medium consists of MS+2.0 mg/l TDZ+ 0.5 mg/l NAA. The medium optimum for the propagation of adventitious buds consists of MS+0.2-0.5mg/l TDZ+0.05 mg/l NAA+100 g/l coconut water extract, and the medium optimum for the rootage of the test-tube seedling was MS+2.0 mg/l IBA2.0+100 g/l coconut water extract.
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Hello everyone
I am working in mango micropropagation through nucellar embryogenesis in Ratna (Mono-embryonic). Among 722 developed plants. I found one off type plant, the morphology of one leaf quite different (but other leaves are normal). During in vitro phase, yellow patches were observed on dorsal side surrounding the midrib, but no out growth was observed on ventral side. But after 2.5 months of hardening period, (total 4.5 month old plan), wheat like of outgrowth was observed ventral side (opposite) of yellow patches. Now gradually it’s turning to brown.
Can anyone suggest the exact cause of this symptom?
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Have you checked the ploidy level? In other dicot species haploids show this phenotype...
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The axillary microshoots/ pseudostem arising from in-vitro grown banana look very thin, although they seem to be healthy and fleshy. The media I am using has full strength MS + MgSo4 + Vitamins + BAP + 3% Sucrose + 0.8% Agar, pH 5.7. What can I do to increase banana shoots' diameter/ girth or to make them grow in a clump?
PS: Picture 2 is how the tissues look like while picture 1 depicts the exact girth I want to have or induce in the banana explant.
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Dear Swati
The cultures were incubated at 25±2ºC under 16-hour light period with light intensity of 2000 lux cool white, fluorescent lights for the growth and development of shoots.
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I am looking to find out if there are any plant species that is a challenge to tissue culture. Whether it is due to lack of a known protocol or it just has never been tried before.
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There are still many plants especilly woody species where micropropation has not been tried. Howerver, let me mention that micropropagation is mainly carried out to resolve a specific problem, and not just for pleasure. So you need to first of all identify a specific problem that natural propation of the plant in question can not solve., like low and lengthy time to produce seeds or propagules, production of disease free propagules etc before proceeding.
.
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I want to try micropropagation of saffron and I search for the best method ; Organogenesis or Somatic Embryogenesis. and how time take each method ?
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Dear Mohammed
In the attached paper, micropropagation of saffron using direct or indirect shoot induction or plantlet regeneration through somatic embryogenesis ha described.
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Dear Friends
I see that many people use plastic boxes for plant micropropagation. Can you please let me know the type of plastic these boxes have to be made of and the method on their sterilization
Thanks all in advance
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Is there anyone working on in vitro propagation of gf677 and GN rootstocks?
what's the best medium (include macro and micro element and Plant growth regulator) for proliferation and rooting?
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Hello. Dear Mohammad I also work on the proliferation of G×N15 rootstocks in Plantform bioreactor. I recommend that you post your poster at the Genetic Congress.
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Dear Friends,
mayby somebody have experiences with in vitro propagation this species? We can disscus together about the medium and plant growth regulators.
Regards
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Dear Agnieszka
The appropriate treatment may vary depending on the species and variety. In a study on two selected varieties of Calathea crotalifera, the highest number of multiple shoots was obtained in MS medium supplemented with 3.5 mg/L BAP, 1.0 mg/L NAA, 3% sucrose, and 6 g/L plant agar for both varieties. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L NAA. In C. ornata cv. Sanderiana, the highest number of shoots was obtained on MS medium containing 2.5 mg/L BAP, 2.5 mg/L Kin, 4.5 % sucrose.
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Plant tissue culture
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With best wishes for all researchers i have a question about micro propagation or direct organogenesis of phalaenopsis micro propagation in tissue culture medium. I cultured the whole plant with small size in MS medium with 3 mg/l BA and 0.5 % activated charcoal. but after 2 mounts there was no any proliferation. is there any one can help me in this problem?
Best regard
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why do you use activated carbon (CA) at this stage! it does not correspond! CA is used in the rooting stage to generate a dark environment, control phenols and induce rhizogenesis. You must prioritize the production of adventitious shoots by trying different concentrations of cytokinins WITHOUT CA.
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Can anyone suggests how to prevent my cultures of plants from getting blackish apical leaves with stunted growth of culture after two sub-culturings? Does anyone know the cause of it and/or cure for it?
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some times it occurs because of calcium or boron deficiency in tissue culture medium. it depends on your plant your explant, your calcium and boron concentration and pH.
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Callus cultures in plant regeneration systems are sometimes useful for conservation. Moreover, they are capable of new variants. What do you think about secondary metabolite production from callus mediated regeneration systems? Is it feasible to try new variants in vitro which may provide more secondary metabolites than mother plants? I think, questions should alternatively be extended over duration of callus cultures, if the new variants are the case. "Have you ever tried/seen or read any study to check metabolite profiling of the regenerants (regenerated plantlets) derived from different callus culture periods?"
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Dear Moniika
·this review helps, on shikonin pigment
Sonia Malik , Shashi Bhushan , Madhu Sharma &Paramvir S Ahuja, (2014):Biotechnological approaches to the production of shikonins: A critical review with recent updates. Critical Reviews in Biotechnology 36(2):1-14,......................Good luck
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Hello fellow plant tissue culture researchers. I am collecting data on vessels and problems in tissue culture labs for my masters thesis. Please fill out this short survey:
Thank you very much!
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When work in tissue culture you must be patient and in accurately work , you must read a lot about your plants and the protocol you should follow. Good luck
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Dear Researchers
I read some paper about micropropagation from hairy root culture and some times the plant micro propagate from hairy root produce dwarf plant. can i use this method in ornamental plant to produce dwarf and smaller plant or produce plant with massive root in Aquatic plants culture?
Best regard
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Dear Yousef
The plant dwarfism caused by someclonal variation has been reported in some plants and can be used for plant breeding.
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I am working on the cultivation of Labisia pumila via tissue techniques. Currently, I'm looking for the best micropropagation system for it. I found a few papers reporting the cultivation of this plant by using Organogenesis. Anyone have any better information on this subject?
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Hi,
Thank you for the paper. Really appreciate it.
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Proliferation of nodal segment excellent on dkw without contamination but after 3 week it died... I try subcultivating every week on new dkw but not good? Maybe after proliferation need some diferent media and hormon concentration... Any help?
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Buhara, that's a nice proposition: shoot is big enough to growths by itself. Condensation might be also a consequence of a reduced exchange, proper of tight closed vessels. In any case, it must be avoided.
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Lovely friends,
I need your help. We are working right now with Gillenia. Micropropagation is working very well but we have problem with acclimatization phase. After planting slowly all plants dided. Have sombody any experience with this special variety?
I am looking for your infos 😉
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Place your vitroplants under the following conditions:
- on a light and draining substrate (peat or peat and perlite),
- in a humid environment (covered by polyethylene or under mist system)
- A substrate temperature close to 25 ° C.
- under shade 50 to 70%
- irrigate the first two weeks with a fertilizing solution
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Greetings to all colleagues... i have some quastion: pH level of my media must be 5.0.... pH level of my media is adjust to 5.0 before autoclaving and I put zeatin after autoclaving and cooling the media below 50"C trough steril siringe filter (zeatin can not be autoclave - is not thermostabil). My stock solution (100ml) of Zeatin is dissolves in 1N NaOH (3ml) so that is the problem, because NaOH increase pH level !!!
I can not adjust the pH after autoclaving because The pH sond and the pH adjustment fluid (1N HCl) are not sterile!!!!
So please how you dissolve zeatin and adjust pH level after autoclaving?
Are you maybe dissole Zeatin with etOh and it is posible et all?
I tried to adjust the pH level (5.0) stock solution of Zeatin with 1N HCl = without success, Zeatin was clotted again
Thank you
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I add zeatin and adjust the pH (5) before autoclaving at V. corymbosum with very good results
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A parasitic plant.
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Hello
Please find this paper.
Becker, H.; Schwarz, G. 1971 In vitro culture of Viscum album L. Z. Pflanzenphysiol. 59:273–278.
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I'm using micropropagation techniques to accelerate the propagation of strawberry
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Hello
I think the fresh nodes or runner segments and runner tips from strawberry mature plants are the best explants for micropropagation of strawberry.
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Has anyone been able to initiate bamboo shoots from nodal cuttings? If so what's the best culture media for D asper? l have failed to achieve shooting with Woody plant media and M&S with BA at different concentrations using field growing explants. The other challenge has been moulds after decontaminating with hypochlorite and ethanol; how can l tell if the fungi is endogenic?
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