Quick transfers to fresh medium may help, but would be labor-intensive.

Polyvinylpyrolidone (PVP) has been incorporated into media with some success for teak cultures. You can Google "PVP medium teak" to find some references.

Be aware that some phenolics actually promote shoot multiplication and rooting. As usual, the dose makes the poison.

you must use anti oxidants solution  for immersion of the explants, such as a mixture of citric acid and ascorbic acid , you could also use PVP, later on.

Dear Abdel salam, Use of activated charcoal powder in the medium can prevent the exudation of phenolic compounds  from the explants cultured in vitro . Wish you good luck

use prefilter mixture of citric and ascorbic acids and add them to autoclaved medium when its temperature can be tolerated by hand  and mix them well in your jars

activated charcoal has some of the best moderating affects

Dear Abdel Salam, I wish to add- If you are convinced  of activated charcoal powder to retain phenolics ensure uniform suspension of charcoal in the medium. To achieve this  stirr the culture flask or tube  with the medium  when they are about to attain room temperature.

To reduce 'browning', different plant species probably has its own preferred way. In cotton tissue culture, to reduce 'browning' caused by phenolic compounds, we used glucose (instead of sucrose) as carbon source, and use 'Phytagel™' as gelling agent (instead of agar). Good results were observed.

One natural way is adapting source tissues to very dim light before taking out tissues. The treatment is found positive in several establishemnts trials. http://www.hort.cornell.edu/uhi/research/articles/HortSci%2022%285%29.pdf

Dear Prof. Dr Abdel Salam , first of the all it is very important question : in the first don't sterilize of explant with colorex but use MC for sterilization, use of silver nitrate in the medium with PVP and reduce the lighting lux , or you can carrying of incubation process under darkness for 3 days and   finally make subculture between 7 to 10 days not more . With my best regards 

You can use Casein Hydrolysate and PVP both or alone

Actually this is a challenging thing that happens especially when dealing with regeneration of  perennial plants like trees. Often recommended to change the medium  (what they call it sub culturing).  This prevents from production of  phenolic cpd or browning of the media.

Dear Sir, based on your description, I would suggest that careful handling of explant materials are important from the very beginning of the sterilization stage.

It would be best to minimize mechanical wounding incurred onto the explant while trimming the edges after disinfecting them.

Rinsing of freshly isolated explants with antioxidants would be another method to delay browning as this would lower the redox potential of solutions.

I hope this is useful and all the best.

Dear Resercher

you could use activated charocol

kind regards

There are a few ideas to solve this problem:

1) cutting under liquid antioxidant mixture (ascorbic acid, citric acid) or destilled water to avoid oxidation during start of culture

2) for establishing of culture is better to use liquid medium (e.g. in tubes with paper bridge) that allowed simple and quick change of medium by Pasteur pipette,

3) on solid medium you can put absorbants e.g. active charcoal or polyvinilpyrolidone (PVP) 

4) browning is caused by oxidation of polyphenolic substances, thet are parts of defense reaction, so conditions at first stage of culture should decrease metabolic processes, I recommend put cutlures to the dark in a fridge at about 4°C

5) defense reaction could be increased by endogenous microorganisms, liquid medium in fridge is a good system for their using and management 

Good luck

There are several ways:

Use of antioxidants in medium

Use of charcoal in medium

Frequent subculturing if there is there is slight response from the explant

incubation of explant in antioxidants like ascorbic acid etc.

Luck....

Dear Abdel-salam Abdel-hamid Reda. Although I have never had this problem, I know that some researchers use activated carbon to adsorbe phenols in culture media. The most common concentration used range from 0,5 to 5 g/L

Quick transfer of explants, dissecting explant tissue under the surface of liquids, incorporation of activated carbon, ascorbic acid or citric acid in the culture medium prevent brownimg of the medium. Try any of these procedures. PVP in the medium has also been effective.

Dear Sir,

Please try followings..

1. Use very sharp scalpel / blade during explant preparation, because injury in the cut edge may cause browning of the explants.

2. Supplementation of any one or combination of antioxidants (PVP, ascorbic acid, citric acid, etc.) in medium.

3. Addition of activated charcoal in medium.

4. Frequent sub-culturing (within 10-15 days), particularly during first 5-6 sub-culturing.

Some authors recommend transferring plants every week, or if it is possible earlier to fresh media. Maybe this  paper could be useful:

Micropropagation Systems of Feijoa (Acca sellowiana (O. Berg) Burret) (2013)

Miguel Pedro Guerra, Gabriela Claudia Cangahuala-Inocente,

Lirio Luiz Dal Vesco, Rosete Pescador, and Clarissa Alves Caprestano . In: Lambardi M, Ozudogru EA, Jain SM (Eds.). Protocols for micropropagation of selected economically-important horticultural plants. Humana Press, New York. 45-63.

the only effective product is activated charcoal

kind regards

active charcoal or polyvinilpyrolidone (PVP) are the most used in medium to avoid the accumulation of phenolic compounds in tissus

Helena has included a number of possible steps that can be tried for preventing browning.  Another simple step that can be used is frequent subculturing to prevent the buildup of the phenolic oxidation products in the media around the cut surface of the explant. Be careful with the use of the absorbants though, it will also absorb your phytohormones from the media, so the effective concentration of available auxins/cytokinins will be lower than what you put in.

Add PVP in the semi solid medium or use liquid medium.At the time of collecting the planting materials from garden use ascorbic acid solution ic, after cutting the stem dip the cut end in Ascorbic acid solution.

Best method for controal is use of active charcoal

Frequent sub culturing is best  because in the beginning of first inoculation of the ex plants phenolics used to exude out, I  hope  after two three frequent sub culturing problems will be solved. Try to remove the browned portions from the explant every time.....if the problem is existing you have to take care of your hormone concentrations , some times if you are using higher concentration of auxin will also damage ex plants making them brown....

It may be olf fashioned, but when taking out explants the first care is lowering polyphenolics by an old, very old technique made for endive and other vegetables. This allows a delayed response to cut and browing. Low light is an essential step for conditioning the plants to takings clear buds. https://books.google.it/books?id=QYcEAAAAQAAJ&pg=PA428&lpg=PA428&dq=bleaching+endive&source=bl&ots=dBSBPhWGbE&sig=_Toy1hi4U-Zr9AgOaiRfa78s4Yk&hl=it&sa=X&ved=0ahUKEwjD6o-43q3JAhWFXRoKHWuhCMAQ6AEIQDAF#v=onepage&q=bleaching%20endive&f=false

Helena Vlašínová and Muhammad H Rahman ·gave good explanation. In my experience, the addition of activated charcoal and subsequent sub culturing sounds good in alleviating the effect of phenolic oxidation (browning).    

Hi all dear

You must control the "PRODUCTION" and release("EXUDATION") of phenolic compound (step by step).

1. Mother plants to put in a dark condition or Cover with a cloth bag.

2. Do not water the day before.

3. use the sharp knife.

4. soaking the explants in the Antioxidant solution.

5. Tap Water for 60 to 120 min.(The Explants will immerse in   NaOCl (5% + 95% Water) for 3 min before T.P. ).

5. sterilization

6. soaking the explants in the Antioxidant solution (again).

7. cutting of end explants.

Lower the pH

Use antioxidants (citric acid, ascorbic acid)

But the most effective traiement is activated charcoal

Adding PVP is better solution

Original PaperPlant Cell Reports

February 2015, Volume 34, Issue 2, pp 291-309

First online: 28 November 2014

Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress

Yinghui DanAffiliated withInstitute for Advanced Learning and ResearchDepartment of Horticulture, Virginia Polytechnic Institute and State UniversityDepartment of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University Email author 

, Song ZhangAffiliated withInstitute for Advanced Learning and Research

, Heng ZhongAffiliated withSyngenta Biotechnology, Inc.

, Hochul YiAffiliated withSyngenta Biotechnology, Inc.

, Manuel B. SainzAffiliated withSyngenta Biotechnology, Inc.

Thanks to everyone who participated in answering my question. Really very usefull to all.

With best regards

Dear prof Abdel-salam

Browning phenomenon can be prevented by one or more of the following besides all mentioned above :-  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

Browning is more frequent in the first estages of the culture. During the first two-three weeks transfer the cultures to fresh media several times to avoid phenol accumulation, also remove the botton end black part of the explant. That will help you to reduce explant losses.

Charcoal powder in the media helps to control phenolic effect in the first stages of the culture. Sometimes we had tried liquid media with a supporting bridge with filter paper. 

I have the same experience as Utpala, together with lower temperature and light, for decreasing of metabolism speed.

quick transfer on fresh media

use of ascorbic acid

add activated charcol

higher conc of Riboflavin

T. Bettaieb ; 

In my opinion, there is only one valid solution: activated charcoal

Best method to control use of activated charcoal

use TIS (temporary immersion system), install a pipe system that may remove the excess air in the tank, and refresh the air inside.

Use semi hard wood shoots as source and antioxidant treatments is useful.

Shoot-tip(meristem apex of shoot) in culture exudes little phenolic components in the medium provided explant source is from juvenile part or plant of woody plants. This is further to my earlier answer.

One of solution use activated charcoal

cutting under liquid antioxidant mixture (ascorbic acid, citric acid) is so important to reduce browning from the start of culture

Ascorbic acid is helpful in reducing browning in culture

We are using activated charchol when observing browning of culture media due to the oxidation of phenolic compounds. It is also advisable to transfer to fresh medium.

Use of charcoal in media and regular transfer of the sample is a good solve

Activated charcoal, antioxidants, as stated above from many colleagues...

Dissecting your plant tissue in ascorbic acid solution / or wash immediately after cuts. It will prevent air contact and browning. dont worry about remaining liquid, your media will take care of it. Using activated carbon with ascorbic acid also works very well.

Place coal or pvp material as well as planting agriculture at close intervals . Minimize iron in the media

Add PVP to the medium

In the detergent stage, the phenolic compounds can be used to prevent the release of substances antioxidant such as citric acid, ascorbic acid, the combination of these two substances, and active charcoal.

You can use anti-ethylene agent such as silver nitrate and antioxidant compounds such as activated charcoal, ascorbic acid, Polyvidone in culture medium. You can also short the time between sub-cultures.

Activated charcoal or antioxidants

Frequent subculture is the best way without the need of the study to optimize the concentration of activated charcoal

Helena Vlašínová Ma'am: How many days or hours should the culture be kept in dark and at 4 degree temperature.

Quick transfer to fresh media or

Use of ascorbic acid in medium or alternatively

Add riboflavin

In my experience of working with cotton in tissue culture, I was getting browning too frequently. Best to protect from phenolics was usage of ascorbic and citric acids together. Plus sugar source matters. Glucose reduced phenolics significantly

Add citric or ascorbic acid each one alone or together, try 100-250mg/L per each. Add them after their sterilization by biofilters to the medium as you tolerate the hot medium container by your hand never sterilize them through the autoclave and shorten the subculture of the plant cultures some times one subculture every one week until phenolics be ended. however, when you use autoclaving for citric or ascorbic, the high temperature will destroy them. also, you must quiet the lights and avoid high temperature in room culture in some time the long dark period work for less or stop physiologically the release of phenolics.

Activated charcoal

Charcoal also is good but maybe making some problems with the root system in vitro, however, use it but not for a long time.

With my experience in different crops Banana, sugarcane, date palm etc, when we do dissect the explants due to oxidation process, explant release poly phenolic oxidase (Quinines) which is brown color. Follow the below procedure to avoid this issues.

1.Addition to your surface sterilization procedure , use Ascorbic acid and citric acid both 100mg per liter for 20 min as antioxidant treatment.

2. This surface sterilized explants need to be transferred to respective media media with Activated charcoal 750mg / liter. try to sub culture this cultures at the earliest (10-14 days). Still problem continue u@@@@se combination of Ascorbic acid 50mg+ Citric Acid 50mg and Activated charcoal 500mg per liter in your respective crop media to avoid phenolic exudation (Quinine) . This problem will be solved after two time consecutive transfer of your cultures . For further info contact me through +91 8973336039, +20 1111750082 [email protected]

Abdel-salam Abdel-hamid Reda

you could add some PVP in your media and can also treat explant with it.

Liquid cultures can be used by supporting explants with paper bridges followed by subculturing in 3-5 days

with supplementation of polyvinylpyrrolidone (PVP40) 200 mg l−1 you might solve the problem.

In my experience, submerging the explants in liquid medium (without agar; before culturing) with 0.5% PVP is very effective in order to reduce the secretion of phenolic compounds into the culture medium. Of course, the addition of anti-phenolic substances in the culture and regular sub-culturing can not be neglected.

PVP is a good compound which helps to reduce the phonology effect of the explant.The media should be prepaired with PVP.

The use of antioxidants before and after surface sterilization of explants may help to reduce the leaching of phenolics in the medium. Even the use of some antioxidants in the medium itself also helps. If still the problem persists subculture the cultures early to the fresh medium.

0.25g of activated charcoal can also be included in the constituents of the plant nutrient medium. This can cause a reduction in the secretion of phenols that can eventually lead to the death of in vitro cultures after they're serially transferred into fresh medium.

Greetings and Regards

Adding activated charcoal, various antioxidants including ascorbic acid, citric acid, vinyl pyrrolidine, ethylene inhibitor, and silver nitrate to the culture medium is often used to counteract the secretion of phenolic compounds.

Abdel-salam Abdel-hamid Reda, Treat explants with antioxidants like citric acid, ascorbic acid etc., and put at low temperature for 20-25 min. Add the same types of antioxidants in the medium also to reduce the oxidation of phenolics. Fast subculturing could be the third and most important step to overcome this issue in tissue culture. You can read our recently published article on the Santalum album for more details.

Use of antioxidants before inoculation and in the medium could solve this issue.

Activated charcoal can be incorporated in the medium. It will help adsorb phenolic exudates release from the culture. Also frequent subculture may also reduce the problem.

Dear Abdel,

You can supplement your media with compounds like cysteine, activated charcoal, PVP, arscorbic acid and citric acid to avoid the problem.

All the best,

Mônica