Two important things about this method confuse me.
1. some of the researcher say ascorbic acid and citric acid are heat sensitive, so cannot be add to MS medium before autoclave.
2. 2nd thing is pH, of medium, if i add these antioxidant after autoclave in laminar flow unit, how can i manage the pH of medium, because it will change the pH of the medium.
I'm no expert, I just have an interest because I make my own liposomal Vit C.
The whole heat/Vit C thing puzzles me. Some would suggest Vit C is damaged by as little as 40C and others suggest up to 70C is ok. One paper (link below) suggests the damage occurs between 30 and 60C where L-AA is converted to DHAA and recommends using higher than 70C.
Do you need to use ascorbic acid or would sodium ascorbate suffice? This would avoid pH changes.
http://www.ncbi.nlm.nih.gov/pubmed/20546391
Citric acid are heat labile, so filter-sterilization should be used. Instead of adding it into medium, researchers have treated the explants directly in the sterilized solution to reduce browning problem. See the paragraph: "Pretreatment to explant tissues prior to culture" at:
[ https://books.google.com/books?id=QiErEvCI9rQC&pg=PA26&lpg=PA26&dq=prepare+citric+acid+for+plant+tissue+culture&source=bl&ots=zbGLy1XuQb&sig=4AkxNug2BubryA-6wONQau2J_iM&hl=en&sa=X&ved=0ahUKEwis_szhhI3LAhVK0mMKHWauAgUQ6AEIJTAC#v=onepage&q=prepare%20citric%20acid%20for%20plant%20tissue%20culture&f=false ]
Also read through this similar discussion on RG, which have some useful info.
https://www.researchgate.net/post/How_do_I_reduce_the_Browning_in_Tissue_culture_media/7
Both Ascorbic and citric acids are heat sensitive. It is recommended to add after autoclave. At present for my research, I am adjusting PH of ascorbic acid and citric acid solution also at 5.8 and adding to final media.
For example, for preparing 1 lit. media, i am autoclaving 950 ml media after adjusting PH at 5.8. Remaining 50 ml containing ascorbic and citric acid added to the above autoclaved media through 0.22 filter sterilization after adjusting PH 5.8.
Dear Ravindera kumar when i added citric acid 20mg/L to autoclaved media in laminar flow unit the media did not solidify, may be the citric acid drooped the pH of media that's why it didn't solidify. so how can i manage this issue.
May i know the purpose of these two anti oxidants for your medium.
If your aim is to prevent the oxidation of polyphenols, you can also try with Poly vinyl pyrrolidone or charcoal.
Hi Yuan
In regards with pretreatment with ascobic acid, if my explant is shoot meristem, would meristem store in ascobic acid?
TY
One can not use ascorbic acid in the culture medium for plant cell/plant.
For the citric acid, it is better to propare stock solution (fresh), adjust pH (K or Na citrate) and add after autoclaving.
ISSN 1061-9348, Journal of Analytical Chemistry, 2007, Vol. 62, No. 8, pp. 742–747. © Pleiades Publishing, Ltd., 2007.
Compounds that are heat-unstable, and thus should not be autoclaved include, ABA, ascorbic acid, cysteine, citric acid, folic acid, gibberellic acid, IAA, IBA, kinetin, nicotinamide, pantothenic acid, pyridoxine, 2-iP, thiamine, and zeatin;
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