if it is due to phenolic or any volatile constituent you can use PVP or activated charcoal in you culture medium...if it does not have any volatile content then it is due to leaching you should try re-cutting the cut ends of the ex-plant.

hi Arunkumar

1. You can use other disinfectants.

2. Reduce the duration and concentration of the disinfectant.

3. Washed with distilled water and autoclaved twice the time to give up.

4. Use of activated charcoal in the medium.

5. Antioxidant substances such as ascorbic acid and ... in the medium use.

good luck

The use of activated charcoal in the medium helps to absorb toxic compounds generated by the plant. Anyway, you have a problem with the phenolization of the explants, in this paper (link) , they cultured in MS supplemented with 50 mg/L citric acid (antioxidant) and they used activated carbon 1g/L. You can try this one and then observe. Sorry for my english.

Try your best, and good luck.

https://docs.google.com/viewer?a=v&q=cache:Ng30ecQffdsJ:www.academicjournals.org/jmpr/pdf/pdf2012/14June/Rodr%25C3%25ADguez-Sahag%25C3%25BAn%2520et%2520al.pdf+&hl=es-419&gl=co&pid=bl&srcid=ADGEESjxmNwlevOx-nwahAc8HQporXudfX4i3S00GlinALJ9v2D_izbY9NA71DRha0_Rom4fVHIDaXhcNGgS1lWlAJX1ilCxqJ1k_ih5W3rlsfqi4dzfEZGxqJyN8mxE-XtoXDKg37Re&sig=AHIEtbSkPY3eGA-Iddw0olfrRe-XaaYHNQ

Ya it is indeed a severe problem. Mr. Ali is right and you can use activated charcoal in a reduced concentration. you can also try with ascorbic acid, citric acid and hydrogen peroxide to avoid browning of explants and culture medea. Last but not the least try to subculture the callus within a minimum passage. Best f luck.

The problems of phenoloic exudation to treated ex plants of antioxidant solution.

All so use in culture media. Antioxidant component ( ascorbic acid , citric acid, and PVP).

Regular sub culture 3-5 days. to reduce the phenoloic problems

yes Mr Durga

Regular sub culture 3-5 days. to reduce the phenoloic problems!!

Hi Arunkumar, which medium have you used and which growth regulators? You should avoid to use chloride and high nitrogen in the medium.This is a main reason of browning because of secondary metabolism has been induced under nutritional stress. Antioxidants will help only a little, because they do not prevent transition of callus to secondary metabolism. Good luck!

Mr. Arun I would suggest that you should check light and dark condition in browning problems.

Please add charcoal to the culture medium. This will absorb the secondary metabolites exudates from the explants thereby avoid browning of the media. Once browning takes place, then it will check the nutrients entering into the explants from the media and affect the proliferation or growth of the explants.

Explants turning brown is totally due to over exposure to disinfectants. You have to standardize the timings of exposing your explants to disinfectants/sterilants/detergents.

The ali gouran suggestion are all right, follow it.

Mr. you do not mention your plant name, nature, and the habitat they grow

You can give treatment of antioxidants like ascorbic and citric acid to your explant before inoculation, you can also add activated charcoal in the media as also suggested by others.

Have you try induction in dark?

The dark can minimaze active phenolic

I will also suggest adding activated charcoal (AC) in the medium, as I have used 0.2% AC for pearl millet shoot induction, as extensive browning were observed and got positive results with use of AC. Agar concentration and incubation temperature will also affects tissue physiology and growth.

You can try PVP in the media in different concentrations, also can try frequent subculture for several times.

You can add a little activated charcoal in the media and switch to use Gelrite, because Gelrite contains no contaminating matters (e.g., phenol

ic compounds) as those found in agar that are

toxic to certain sensitive tissue.

Although it is not that clear what kind of plant material you are working with and how soon the browning occurs I may suggest the following:

- The browning of the medium occurs due to the interaction between the medium itself and the explant plant material. The explants plant material may provide some exudates to the medium. Those exudates may contain high concentration of polyphenols and the browning which occurs is actually the oxidization of phenolic compounds.

- The other possibility that the exudates may facilitate the oxidization process of sugar and organics in the medium.

- Henceforth, you need to establish whether the problem is case specific, meaning does it occur only with you plant material? For that try to use some other not relevant plant material.

- If it is proven that the problem is case specific, you may want to experiment with ascorbic acid and charcoal as described in several previews comments.

- The medium composition, you may want to reduce the concentration of organics and sugars in it.

Ta-ta

Browning is generally due to the leaching of phenolics from the explant which are generally old and mature. To check browning we add adsorbents such as activated charcoal to the tissue culture media. We can also supplement our media with antioxidants to check browning.

To avoid browninig 100 % from your initial culture or duringsuccessive callus development-

1- Use of antioxidant (ascorbic acid and citric acid) @ the rate of 50-70 mg/l in the medium

2- If the browning is exessive, add activated charcoal along with anti oxidants

3- Incubate culture in dark

4- If there is a chance for browning of tissue during subculture, add antioxidants -rate is mentioned above.

You can use also reduced glutathione (GSH) as antioxidant compound

all suggestions are good. If you are adding activated charcoal you have to be careful about your organic additives such as growth regulators, vitamins etc. This is the proven method (adding charcoal) in orchid and perennial woody plant tissue culture. Sometimes, activated charcoal also adsorbs some of it and reduce the available concentration. Adding a bit more ascorbic acid is good, but it changes the pH of the medium. So check the pH after adding it. You can also try more frequent subculturing, however this is more costly. So you need to do a quick cost benefit analysis. If you want to save your culture regardless of the cost go for frequent sub culturing.

I work on woody plants. Sometime we start maintatining the cultures in darkness condition for few days (1-7) to avoid (or limit) the tissues deterioration.

For chickpea i tried all these suggestions but the problem is toresolved i changed the hormonal balance the cultured was good at the begining but now i am confronted to callus necrosis

I agree with Pavan Wardak in order to use antioxidant compounds. You could add GSH (100 mg/l) to the nutritive substrata.

Activated charcoal and ascorbic acid would be the best choices.

Please, also remember that ascorbic acid is very unstable in the culture medium, so, of you have seen some effect, it may rather related with products of ascorbate conversion, but not with ascorbic acid itself.

At least you should prepare ascorbic acid fresh each time.

For the GSH it is not so simple. There is quite a lot side effects. for details you can look Potters et al., 2010.

http://link.springer.com/article/10.1007/s11627-009-9266-y

Good luck!

My past experiences on the use of GSH were significant to limit the peroxidase activity and reduce the hyperidricity on shoots of apple proliferated on liquid media.

Hi, Maurizio, you are completely right, GSH have a lot positive effects on plants, but these effects do not related with antioxidant activities.

Media browning is because of releasing of phenolic compounds from the cut edges of the plants. You must prefer activated charcoal in the tissue culture media or you may use Polyvinyl pyrilidone (PVP) in the medium.

You can add activated charcoal and l-ascorbic acid to overcome this phenolic compounds and good luck

Add activated 1% charcoal or Poly venyl pyrrolidone in low amount.

I suggest the use of PVP or actived charcoal. Try to test also the light source, taking in consideration a short period in darkness conditions

You can use ascorbic acid with citric acid or pvp in little amounts. This only use if you don't need

to produce phenolic compounds as secondary metabolites.

Sealing the cut end of explant with paraffin wax has been beneficial in controlling browning by preventing exudation in Dioscorea alata.

For details Refer Bhat SR and Chandel 1991. Plant Cell Report 10: 358-361

Excellence is Doing an ordinary thing in an extraordinary way

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If we add a para daily after one month it will result in a productive item (paper , review, thesis , chapter, etc.etc.) So start from today

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I am pleased to inform u , after successful publication of two books i have started compiling another one or two books depending upon response from authors and dedicated scientists like you) in the aspect of crop improvement

The themes are

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2. Plant bioticjnology aspects of crop improvement( biotic, abiotic stress, quality improvement,) both Marker assisted selection AND TRANSGENIC APPROACH/ CISGENIC APPROACH/ other in vitro techniques etc.)

3. Molecular breeding/QTLS biotic, abiotic stress, quality improvement

4 Plant genetic resources , conservation and its application in crop improvement

5. Crop specific breeding approaches like rice , wheat , maize or other horticultural crops

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Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

--

*

******************************

*****************************************************

Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

http://www.skuastkashmir.a

Excellence is Doing an ordinary thing in an extraordinary way

"Some thing is better than nothing"

If we add a para daily after one month it will result in a productive item (paper , review, thesis , chapter, etc.etc.) So start from today

Dear All

I am pleased to inform u , after successful publication of two books i have started compiling another one or two books depending upon response from authors and dedicated scientists like you) in the aspect of crop improvement

The themes are

1. plant breeding in relation to crop improvement( biotic, abiotic stress, quality improvement, etc)

2. Plant bioticjnology aspects of crop improvement( biotic, abiotic stress, quality improvement,) both Marker assisted selection AND TRANSGENIC APPROACH/ CISGENIC APPROACH/ other in vitro techniques etc.)

3. Molecular breeding/QTLS biotic, abiotic stress, quality improvement

4 Plant genetic resources , conservation and its application in crop improvement

5. Crop specific breeding approaches like rice , wheat , maize or other horticultural crops

6. Hybrid technology and status of hybrid breeding in various crop plants

7. Molecular farming

8. Plant plastid/chloroplast engineering

9.Genome sequencing of major crop plants

10. other aspects related to crop improvement

Interested scientists , students are requested to send in their titles and abstracts from the above themes before 30 September 2013 for finalization. Selection of papers will be on first come first serve basis.

Interested scientists who want to be co editors can also send in their interest

For any further query kindly contact

[email protected]

shabirhwani@[email protected]

Kindest regards

--

*

******************************

*****************************************************

Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

--

*

******************************

*****************************************************

Thanks & Regards

Shabir Hussain Wani Ph.D, F.S.P.R

Assistant Professor

AICRP NSP (Crops), Division of Plant Breeding and Genetics

SKUAST-K, Shalimar, Srinagar, Jammu and Kashmir, India 190025

Mobile : +91 9419035566/ +91 9797001791

http://www.skuastkashmir.a

Sir you used anti browning/ anto oxident substance and frequently sub culture

If it is the ex-plant which gets brown then it is because of the plant constituents leaching out from the cut ends in to the washing sources such as mercuric chloride or ethanol. This happens because the cell's at the cut ends are prone to loose these necessary constituents. The browning actually creates a wall which does not allow the washing chemicals nor the nutrients to enter the other cells. Thus the ex-plant does not grow. You can avoid browning of the inoculated ex-plant by increasing he length of the ex-plant and re-cutting the cut ends which are already brown before inoculating them in the media.

A good effect on browning is related also with other antooxidant compunds: citric acid or ascorbic acid

Hello,

this is problem if you work to plants with phenols compositions, You can use weak detergent with antioxidant (Ascorbic acid, Citric acid0.

good luck

if it is due to phenolic or any volatile constituent you can use PVP or activated charcoal in you culture medium...if it does not have any volatile content then it is due to leaching you should try re-cutting the cut ends of the ex-plant.

Browning occurs primarily because of the release of the activated charcoals.U can add activated charcoal 0.25% in the medium.Active charcoal acts as a potent absorbant. I have found it very very effective in case of Orchids. Also you can refer this article http://www.springerlink.com/index/j761u81988g63057.pdf

and http://www.sciencedirect.com/science/article/pii/S0734975008000864

Browning occurs due to release of compunds into medium.If your plant is secondary metabollite enriched this will happen. So, if you add active charcoal it will absorb the leeching entities and will reduce the browning.I have mentioned about two research papers. If you go through it , it will help you out.

good luck

Paromik

Browning is mainly caused by secondary metabolites like phenolic compounds and also associated with oxidation in explant. To control browning frequent sub-culturing can be helpful and also used ascorbic acid, casein hydolyzate and activated charcoal in media to get rid of browning.

Use antioxidnats (Ascorbic acid + citric acid & PVP soluble) soltion, either dip in the soltuion or add it in themedia depends up on your convenience and response of your explants.

Pvp or activated charcoal

Use of ascorbic acid (100mg/l) in the medium was beneficial in Tylophora indica

Age of your explants also matters. try to use tender explants and transfer the explants to fresh media regularly.

Hi,

I had such problem when I was using inter node of guava (Psidium guajava) which has high level of phenolic compounds, I tried several antioxidants in the medium, activated charcoal, etc. no way. I solved the problem by coating the cut ends by commercial silicon and that was the ideal solution with more than 99% survives. you can see my article: Improvement Psidium guajava using micropropagation.

Regards

To avoid the brownish colour formation, it is suggested to use PVP or animal charcoal in the media

Try powdery activated charcoal- it's playing well in grapevine and citrus tissue culture

Try activated charcoal. It's work well for grapevine and citrus tissue culture

Phenolic compounds are the major inhibitor of an efficient tissue culture method. If your plant is rich in phenolics then you need to use PVP or activated charcoal in a low percentage. If this is not working then you have to use liquid culture medium with very gentle shake using a shaker usually 100 rpm works as rigorous shaking may break callus. While using PVP or activated charcoal also you can reduce the percentage of agar and frequent subculture sometimes helps in get rid of browning.

Use as orbit acid and /or citric acid in the medium. If still the problem persist add activated charcoal as well in the medium.

Maybe:

Oxidation of tissue - deal with antioxidant by adding to the culture medium 0.1% polyvinylpyrrolidone or 0.3% activated charcoal

Use ascorbic acid 500mg/l....or citric acid......sorry for misspelling above...

Manipulating the explant prevent the blade of the knife is not too hot not to damage the tissue

You can reduce the browning in TC by avoiding TC contamination as can as possible through working in the antiseptic conditions.

you can reduce the browning by the addition of PVP or any antioxidant like Ascorbic acid or Citric acid

Hi dear

For decreasing the browning in tissues cultured in vitro there are some scientific ways suggested in book related to subject:

For example: you must avoid any extra and unneeded cut or injury in tissue, you can add activated charcoal to medium, you can control the light condition of cultures (maybe about some cultures you can cover your glass jars with aluminium foil, you must keep the tissues young and powerful (some old tissues or some shell and crisp tissues are capable for browning, especially about calus you must keep them in a good condition), you must notice to part of plant that use for in vitro culture (some plant parts produce crisp and notpowerful callus that are capable for browning, that this can be deducted with experiencing with different parts of that specific species that you use…)

Anyway, you are the best person for finding the way for solving the problem, because your prescription is specific for your material and can be reached with experiencing.

Best wishes

Serial transfer is another way to reduce browning besides adding some adsorbents in the medium. Please see for details:

Micropropagation of Indian wild strawberry

Indra D. Bhatt, Uppeandra Dhar

Plant Cell Tissue and Organ Culture 01/2000; 60(2):83-88. ·

All these suggestions are good, the combination of them is the good idea, so I recapitulate:

1/ prevention of phenolic compounds excretion (in woody plant I use over mentioned methods culture for the first 7 days at 4°C in dark to decrease secundary metabolite production, also terrestrial european orchids is better to keep after transfer to fresh medium in fridge for about two weeks),

2/ the phenolic compoud removing (I use liquid medium with filter paper support for first days, then regular transfer on gelrite solidified medium with adsorbants PVP or AC)

3/ avoid oxidation of polyphenolic compound (cutting under antioxidant mixture or sterile destilled watter or medium, use antioxidants in medium, dark and low temperature also decrease oxidation) .

I wish you all the healthy (lot of browning is connected wit contamination :() and good growing cultures. Good luck

Wash your material with ascorbic acid (deep for 5 min) and add KNO3 instead ammonium nitrate and reduce it concentration gradually and find out adequate one. this will help you, I think.

Repeated sub culture reduce the accumulation of secondary metabolites in the medium to the toxic level as those are only secreted through the cut surface. Use of ascorbic acid in washing the explant could reduce the browning of tissues. How ever reduced level of nitrates in the medium I found some times lower the growth of tissue

browning can be reduce by the addition of PVP or antioxidant like Ascorbic acid or Citric acid, frequent sub culturing, using activated charcoal

Firstly use juvenile explants for culture. You can also use antioxidants

like- PVP, Ascorbic acid, Activated Charcoal. Frequent subculture of

explants on to the fresh culture media and putting them in dark and at

low temperature can be effective for successful establishment of plants in

vitro and encountering phenolic browning.

In our lab, we have found the addition of silver nitrate (an inhibitor of ethylene biosynthesis) to the medium quite useful; explore with ranges 1 to 10 micromole per litre though even upto 50 micromoles per litre works. Duchefa Biochemie of Netherlands, makers of plant tissue culture medium, also suggest mixing silver nitrate with silver thiosulphate (slowly add 20 ml of 0.1M silver nitrate to 80 ml of 0.1M sodium thiosulphate). The resulting silver thiosulphate complex is a very potent inhibitor of ethylene biosynthesis and will help reduce general tissue senescence and browning.

Erratum: It is actually mixing of silver nitrate with sodium thiosulphate.

Use of oxidized glutathione in conjunction with ascorbic acid to prevent browning of tissues.

Use of antioxidant mixture (ascorbic acid 0.1 g/l + citric acid 0.15 g/l) to reduce browning of plant tissue from phenolics during explants superficial disinfection and use ascorbic acid and/or activated charcoal on the initiation culture media. Also the first week putting in darkness and progressive increase the light. Frequently change the culture medium

we can reduce the browning of medium by the by the addition of activated charcoal , an amorphous form of carbon. The chemical nature of amorphous carbon, combined with high surface area and porosity, makes it an ideal medium for culturing.

Second one is the addition of antibiotic to culture medium.

Third one is the addition of antioxidant. An antioxidant is an electron donor which inhibits the action of labile substances with high schiometeric efficiency. Acrobic acid and citic acid is commonly used in plant tissue culture.

using PVP OR ACTIVATED CHARCOAL OR antioxidant (citric and scorbic acid 

You can use PVP or reduced glutathione (each 50-200 mgs/L) as antioxidants. Also citric acid or ascorbic acid are the same.

Browning phenomenon can be prevented by one or more of the following :- the addition of active charcoal (0.2 – 3.0% w/v) .-  The addition of  pvp (250 – 1000 mg/l ) . -Addition of so-called anti-oxidants (citric acid, ascorbic acid, thiourea or l-cystin). -The addition diethyl-dithiocarbonate( DIECA ,2g/l) in the rinses after of sterilization. -The addition of amino acids (glutamine, arginine and asparagine.

-Use liquid medium to dilute toxic products. -Frequent subculturing onto a fresh medium.  - Keep the shoot bases in darkness (by painting, aluminium foil,

-A reduction of wounded tissue. - A reduction of the salt concentration in the medium.

best regards

CA+Ascorb. Acid50_100mg/l

Pvp 250mg/l

Keep your jars in low temperature for 3 days

Darkness for 3 days 

Imbibe in CA+AS

Cut explants before tab water(first stage)

It would be interesting to inform you in which species this problem is occurring so that we can give a more consistent suggestion

I am agree with Dr. Muslim 

Soak the leaves in ascorbic acid and citric acid solution (25-40 mg/l each) for 10-30 minutes depending upon the exudation of phenol during callusing. Use the same fresh  solution to soak for 5-10 minutes before inoculation.

Use 100 - 1000 mg of activated charcoal to the media for 1-2 subculture and gradually omit the charcoal.

Use mixture of (ascorbic acid 0.1 g/l + citric acid 0.15 g/l) to reduce browning of plant tissue

Hello

Please read the following chapter.

Use mixture of (ascorbic acid 0.1 g/l + citric acid 0.15 g/l) under dark condition

Use a combination of ascorbic acid and citric acid (50-75 mg/l depending upon the severity of phenol exudation from culture to medium along with activated charcoal 100 to 150 mg/l under dark incubation for initial phases (at least up to 2 subculture). Subsequent incubation under 12 h photo period with and without activated charcoal will be useful to control browning.

Hi sir

treat the explant with activated charcoaal or vitamin c or amend the media with activated charcoal

Use activated charcoal

The best way to remove browning is to prevent it by optimized explants metabolism. Here is authors described some key points: In Vitro Cell. Dev. Biol.—Plant 38:116–124, March–April 2002

I think dark conditions and use active charcoal

If aim of the researcher is a callus biomass, dark is preferable. In this case ROS production is much lower. But the key in the preventing browning is metabolism adjusted for callus. Ea. optimal nutrients ratio, optimal hormone concentration etc.

Browning normally exeduces from the stem of the sample, e.g. the stem. You cant "cure" it by transfer every week. Thus try to use another explent. E.g. a leaf though this too might show browing. But you could start right away to add growth hormones and maybe even not to add charcoal as it lessens the amaont of the available hormones. Back then I should have tried it.

Subculturing can be done frequently to reduce growing during culture

Browning is cause by the accumulation and subsequent oxidation of phenolic compounds in the tissue and culture media. Changing the basal media composition, concentration, type of plant growth regulators, frequent subcultures can also be done to reduce the browning. Pre-treating the explants with ascorbic acid, melatonin, and/or citric acid can also reduce oxidative stress and prevent oxidation of phenolic compounds. Another technique is by adding PVPP or activated charcoal into the media and mixed well, then incubate the callus cultures under dark condition.

Browning is mainly caused by phenolic compounds oxidation in the tissues and culture media, and may be don't over autoclaving also some times.

strategies I suggest to prevent the browning of the explant culture are:

1. repeated transfer of the explant to the fresh medium

2. absorption onto the PVP/ activated charcoal

3. modification of redox potential:

addition of reducing agents in the medium/ or the in washing solutions- Ascorbic acid, citric acid, cysteine, HCl, glutathione

4.oxidase enzyme inhibition

5. short term omission of copper-- using chelating agents viz., EDTA, Diethyldithiocarbamate

6.Incubation in dark

Dear Kirankumar Nalla, EDTA chelated all divalent cations, and tossue simple stop growth. Moreover, EDTA is effectively chelated at higher pH (5,5 pH is not optimal for chelating).

Moreover, you can not add acsorbate because they convert to lactones in 3 hours and you will have effect of lactones.

My best wishes!

I often made the subculture when recognizing the browning of explants, but, in some cases for economical purposes, just picked and put the explants to another vacant place in the same culture vessel.

The use of activated charcoal may be a good alternative.

You can also fold a piece of filter paper standing inside a test tube (2 - 3 cm in diameter), with a small hole on the surface of the paper stand, then put your stem or petiole through the hole. The lower part of your explant will be in the liquid medium. The browning compounds will gradually precipitate to the bottom of the test tube.

Try all above. If not, use a new blade, do parafilm untightly or transfer the tissue magenta box, place it under dim light for a week.