I have callus of several rose varieties (6 cutflower variety , 1 miniature and 1 garden variety) but I don't know why they turn brown. The medium and callus is still fresh and the explants are not old and are cultured in 16*100mm test tubes. The medium is Full MS+ Vitamins and NAA with BAP. Can I reduce or prevent callus browning in half MS medium? I will increase callus size and then use it to regenerate shoots .
using Vitamin C, activated charcoal and so on.
a useful way can be subcultureing immediately, such as daily or lower...
so for rose I think u can find more references for this subject.
Thank you Sajede , i will add Vitamin C . subcultureing is not good idea for my situation because contamination rate will be high .
Perhaps PVP-360 (100-1000 mg/l) may help. Reduce Sucrose to 2%; also sorbitol (1000 mg/l) can be useful).
Activated charcoal can be a good idea. browning must come from toxic exudates of the callus. Other possibility is to increase the volume of the media, so the toxic substrances will be in lower concentration
U may add riboflavin or ascorbic acid in the medium!to prevent browning
If your goal is regeneration, browning of the callus may not be a problem. Several species, I do not know about Rose specifically, will regenerate while producing polyphenols. Browning per se is not necessarily a problem.
Browning of callus may be due to oxidation to prevent oxidation you can add antioxidant (Ascorbic in PPM range) in MS medium.
We faced the same problem in cotton and overcame by just using maltose instead of sucrose,. Further using phytagel/gelrite can reduce the browning
R. Kumria · V. G. Sunnichan · D. K. Das · S. K. Gupta ·
V. S. Reddy · R. K. Bhatnagar · S. Leelavathi
High-frequency somatic embryo production
and maturation into normal plants
in cotton (Gossypium hirsutum) through metabolic stress
Plant Cell Rep (2003) 21:635–639
Maybe you can put them in the dark to prevent photo-oxidaton?
Just a small remark concerning the regeneration; do not wait to long to test the regeneration capacity of the callus. If it is non-morphogenic you will never be able to regenerate someting out of it irrespective of what you try (and you loose a lot of valuable time)
It may be due to release of phenolic compounds during invitroculture. Use adsorbent.
Thank you for all comments ,
All explants are in dark treatment , with 30g sugar , callus turn brown when cells come out of thick Rose Petals.
i think that i have to make a new medium with lower sugar and with Vitamin C .
When callus induction occurs, browning can be frequently observed, especially in woody species. Ascorbic acid and/or activated charcoal may be useful, but sometimes not enough. However at subsequent callus passages, apparently healthy tissues can be carefully selected. Try to avoid cutting calli into too small pieces at subculture, since mechanical stress induces the production of polyphenolics.
I agree with the recommendations of use of antioxidants and other medium supplements. But from my own experience with hardwood tree tissue culture, I would suggest you to use reduced dose/concentration of TDZ (Thidiazuron) (0.5 micro Molar) or you can also add to it BAP (2.22 micro molar) to get a very good compact white or greenish callus with high regeneration capacity.
http://nopr.niscair.res.in/bitstream/123456789/19285/1/IJBT%2012%282%29%20265-268.pdf
If you get a result...cite the article. Best wishes :-)
Frequent subcultures to fresh medium and adding antioxidants might help. Sometimes agar/solidifying agent quality and quantity (low concentration) might also affect the callus. Browning occurs at the edges then above factors might be the cause. If the whole callus is becoming brown then the media composition (strength) can be adjusted to avoid this problem.
Hi Mohammed, the browning liekly is due to polyphenol oxidation. This can be prevented by soaking the explants in a solution of ascorbic acid (vitamin C, an antioxidant). The concentrations mus be found empirically as there might be genotypic differences.
Cheers,
HJJ
use 20g/L sucrose, use AC (0.5g/L), replace NAA by TDZ. After callus forming embryos directly transfer your explants to regeneration medium. For callus conservation use 1/2 MS medium with low concentration of Auxins and Cytokinesis .
Hi Mohammed, We had similar problems with grape callus. In switching from Phytagar (10g/L) to Phytagel (2.5g/L), the browning was drastically less.
By adding charcoal powder to the medium browning of callus can be prevented
With my experience in coconut leaf tissue culture, charcoal in the medium will control the browning of callus to a great extend.
There are fiew suggestions to encounter your problem , you can try any one of these-
before inoculation treat explants with 1% ascorbic acid solution or add same to the culture medium.
You can avoid browning by initial incubation of your culture tubes in dark.
My dear colleague,
You can use active charcoal powder(tissue culture grade) and if you store the tubes for long time, you must use low concentration of the basal medium about to half. Antioxidants same as ascorbic acid also reduce callus browning speed.
Thank you , I have Tissue culture grade Activated Charcoal but Activated Charcoal is Risky because i have different results when i use Activated Charcoal in tissue culture so i amnot interested to use it in my roses i think that Antioxidants are more promissing .
Wash the leaf in running water over night or use charcoal in the media.
You can use anti oxidants like ascorbic acid, PVP or activated charcoal in the medium to prevent browning.
Subculture the callus once in a week.... By this we can reduce browning and increase the callus biomass...
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