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.
Questions related to Micropropagation
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.
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.
needed to find resources about using best medium (MS or MT) for Citrus micropropagation
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!
Various plant growth regulators were found to have a positive effect on organogenesis and somatic embryogenesis, but which one is more important?
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?
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.
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.
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.
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.
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.
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.
Non-destructive ways to measure (quantify) micropropagation "success" in woody plant tissue culture
Can turmeric be used as a substitute for the other comercially availble antifungal agents during plant tissue culture?
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?
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
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?)
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
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?
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.
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.
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?
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.
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.
i would want to also know the uses of such chemicals and regents
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
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?
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?
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.
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?
Analyzing different concentrations and combinations of growth hormones on axillary bud proliferation, elongation and rooting
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
For Rosa damascene micropropagation, Use of Which Growth Regulators is to Increase the Number of Shoots Extracted from the Stem Node?
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.
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.
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 ?
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?
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.
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?
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.
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
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.
Is the temporary immersion system used in the production of zamifulia? What kind of system is useful for micropropagation ?
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.
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.
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?
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.
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.
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
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?
mayby somebody have experiences with in vitro propagation this species? We can disscus together about the medium and plant growth regulators.
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?
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?
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?"
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!
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?
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?
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 😉
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
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?