Thanks for your answer. I’ve tried but it didn’t help!

If the browning is due to leaching of phenols then activated charcoal should work, if it is not working then the problem might be aging of tissues?

Have you tried adding some source of antioxidant (citric /ascorbic acid) to the growth media? Alternately you can soak your explants in antioxidants before inoculation.

Hi, Gaia, MS medoium is not suitable for sugarbeet, you should used at least B5 or even better medium. Just one example: MS contain 8 mM chloride, which is micronutrients, and should present in micromolar concentration. There are many other non-optimal ratios between nutrients like very high Zn contents and Zn:Cu ratio etc. So, It is not suprisingly that activated charcoal did not help, as well as antioxidants will not help as well. Good luck! .

Hola, disculpa por escribir en español. Trabajando con Psidium cattleianum tuve problema similar. Realizando al explante, un enjuague con acido ascorbico (5g.L) antes de ser inoculado, redujo la oxidación. Lo que también debe considerarse es que debe evitar exceso de tiempo con el material expuesto a la luz.

Thanks everybody. I think that the age of the explants it is not a problem because I use 3-4 days seedlings that I obtain from sterile seeds.

I will try with ascorbic acid, any idea of suitable concentration?

Thank you very much for your answer.

I used 5g.L as pre-treatment for a few minutes. Evita mantener los explantes bajo la luz, ya que esta promueve la oxidación, good luck!

First thing to understand is the reason for browning? If it is due to leaching of phenolics, then you may try giving a treatment of chilled antioxidant solution containing 100 mg/l each of ascorbic acid and citric acid cooled at 4 degree celcius. Dip your explant in the solction for 5 minutes before inoculating. Also a faster sub-culture cycle can prevent the problem. Try it and see what works best.

I think that the browning phenomenon is due to the production of phenolic compounds, otherwise I don’t know why the explants became brown after 2-3 weeks on the medium. Do you think that the pH could also have any effect?

Anyway since tomorrow I will try the dipping of the explants with antioxidants as you mentioned.

Thank you very much for your suggestions I will tell you if it works.

Hi, Gaia, you are completely right: browning come because of phenolic compounds.

It maen that cells metabolism is far from optimal because of high nutrients stress, and therefore, accumulation of reactive oxygen species. In this case phenolic compounds sreve as as scavenger of ROS. To avoid accumulation of phenolic compounds ypou should normalize plant metabolism by using optimal ratio of the nutrients, and of course, exclude 7th macronutrient (Cl) : because Cl is micronutrient.

Good luck!

Dear Gaia; I strongly recomend you to use AgNO3 in the media.It inhibits the ethylene production. You can start with 1,8 mg/L, added after autoclaving, filtered. I have another idea using ion bombardment, If you want to try it. write me.

I agree with Juan, AgNO3 is very effective inhibitor of ethylene. However, in my opinion it will be much more effective to prevent ethylene production and ROS generation by just normalization of growth conditions, ea. have more cofactors of the Cu-Zn SOD like copper, and more phosphate as co-factor of majority of importnat reactions, and of course, low chloride (100 µM is more than enough). Good luck!

Hi everybody I've followed your suggestions. I treated the leaf explants with ascorbic acid (1mM) and I added AgNO3 (1.8mg/L) to the culture medium. I hope that these will work…

Have you ever tried to add polyvinylpyrrolidone (PVPP) to your medium, to enhance callus formation and reduce browning?

Thanks!

Hi, Gaja, thanks for information. How long was ascorbate treatments? I hope yopu have read Potters et al., 2010 about ascorbate effect of cell culture and consider ascornate stability as well. Have you tried B5 medium? Good luck!

Hi everybody!

I just want to thank you for your suggestions. Thanks to you my calli are white and friable! I just added AgNO3 10 uM in the medium and dipped leaf discs in ascorbate 1mM for 5 min at 4°C!

Thanks again!

One of the probable reason for browning is elevated production of ROS under stress to combat oxidative damage. Production of phenols resulting in browning of the medium and explant. Use activated charcoal, PVP, antioxidants such as Citric acid, ascorbic acid and at cretain extant proline to the medium. Frequest subculture of explants. And keep cultures in dark for 2 week will help you :)

Best wishes

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.

For browing in the cultures -you can use antioxidants such as cu=ystein HCL, Ascorbic acid, Activitivated charcoal among others. Frequent sub-cultures also help reduce the browning

use , PVP, Ascorbic acids do reduce the browning