Dear Lidia Samarina as far as I know there's not described in literature any procedure to clean, or to remove, endophytics. The reemergence of endophytics in plan TC is of great concern for the routine in a micropropagation lab. However, I consider that the most important question is, why reemergence of endophytics occurs? Many factors have been described causing endophytics releasing. Most of them are more or less controllable. However, the greatest impact is produced when aseptic procedures have been strictly followed, and even so endophytics say "hello". In this case, the breakdown of a hypothetical "equilibrium" might cause the appearing of endophytics. Normally after drastic changes of temperatures, endophytics might arise. Continuous checking of the execution of your protocol might prevent the appearing of endophytics.

Really endophyte fungus is very hard to eliminate. First you have to make a rigurous desinfection, after it under, stereoscopic conditions you may take the meristem tissue without leaves (any) and put in the media for growing purpuses.

It is important to identify the fungus you will eliminate.

Dear Lidia, it would be important to know what species of fungus is causing micropropagation problems. One would think that an endophytic fungus should not be so harmful to the growing explant since the vast majority of plants maintain some type of symbiosis, even many times they are inoculated articifially in nurseries, but it seems that this is not the case for tissue culture.

Dear colleagues,

thank you very much for your responses.

However, I will appreciate it very much if you can share the step-by-step protocol of shoots pretreatment which you successfully applied to initiate in vitro shoots culture of perennial/tree crops.

What kind of chemicals (antibiotics/fungicides)?, what concentrations?, how long exposed?, and so on.

I agree that it is important to know the species of fungus inside the shoots, but as you may know some chemicals work against a broad range of fungi. So I hope somebody can share the successful protocol of decontamination.

Thank you very much in advance,

Kind regards,

Lidia

I think first you have to identify which type of fungal contamination you are getting which will help you in confirming the fungicide.

Takling about braod spectrum fungicide, generally bavistin (Carbendazim) which you also came across in many references. The concentration can be used in 0.01-0.05% for 4-5 min.

All the best

Meristem culture of shoot tip or shoot bud of plants at seedling stage in my opinion should give rise to in vitro plant Free of pathogens.

Such plants may then be transplanted to soil following normal hardening and transfer to soil as per standard procedure followed n plant tissue culture.

Dear Dr Razdan,

Did you have successful meristem Culture of TREE species? If yes, could you please write here the protocol of explant decontamination?

Kind regards,

Lidia

Read our research paper:

In vitro clonal propagation of adult tree of neem (Azadirachta indica) by forced axillary branching. Plant Science 166 (2) 501-506; 2004.

You may just pick the meristems and cultivate them. Maybe bacteriostatic substances or antibiotics but with fungi.

I agree with the views on the identification of fungi. But in general, there are two types of pollution (Exogenous and endogenous fungal contaminants). Exogenous fungal pathogens are easy to eliminate from the stock plants using many fungicides such as systemic fungicides (e.g. Benomyl) or other disinfectants (sodium hypochlorite). Endogenous or endophytic fungi become pathogenic to the host plants when the plants are stressed, for example, when the cell walls are weakened or under other unfavorable in vitro conditions. Mature stock plants are highly loaded with fungal pathogens and spores, but used due to their maturity, especially in fruit trees. Overall, for further guidance, some of these chemicals include: 1. Nystatin (toxic to fungi ); 2. Copper oxychloride (fungicide ); 3. Sodium & calcium hypochlorite; 4. Formaldehyde (aldehyde); 5. Benomyl and Captain; 6. Teepol (detergent). 7. Silver nanoparticles; 8. Titanium dioxide (TiO2); 9. Ampicillin 10. Alcohol and so on

I hope the content has been effective and useful.

With luck

Of course, the concentration and duration of treatment in each plant is different and, since there is no information about the type of plant you are studying, it is necessary to search articles of fungicides [available for you] for reaching a sterilization protocol. In my experience, Silver nanoparticles is successful at 200 ppm concentration for 10 minutes. Also, I had a successful experience in this treatment [Sodium hypochlorite (20% with TWEEN® 20; 20 min)+ Alcohol (70%; 60 second)+ twice washing with sterille-distilled water (3 and 5 min, respectively)].

Wish you luck

Reza Ghahremani

Email: [email protected]

Dear @Lidia Samarina, as suggested by others, there's no specific report or protocol to control fungal contamination therefore we need to standardize the sterilization with different treatments and exposure time. Prolong exposure to disinfectant may also affect the growth of explant. In my experience, bamboo is one such plant where fungal contamination is quite common. During the sterilization, washing with tap water or autoclaved water at first step was avoided as it helps in the dispersal of fungal spore. Instead, the explant was wiped with 70% alcohol followed by treatment with 0.5-1.0% HgCl2. This treatment should be followed by proper washing (4-5 times) with sterile water. Further, treatment with Bavistin will also be helpful in eliminating the fungus. Both ends of the explant,as I was using internode, were trimmed before inoculation to the media.

If none of the protocol works thermotherapy of the explant can be tried.

All the best 👍.

Dear Leimapokpam Tikendra,

thank you for the useful advices they are really can be helpful !

we always wash our explants in the tap water for 30 min. maybe it really cause the dispersal of the fungi.

Also we wil try to find fungicide that you mention.

If you need to clean endophytes fungus from shoot, should be better if you make stong sterilitation of your explants and then, under laminar flow, you can go to shoots, find the meristem, really meristem (dome) and transfer to the media.

How meristem tissue is free of vascular tissue, you transfer only cells-meristematic for plant regeneration.

All the best,

Dear colleagues,

thank you very much for your responses I really appreciate it.

Several of your responses advice us to pick out a meristem which is free of infection and initiate tissue culture from the meristem (M. K. Razdan, M Angeles Zorrilla Lopez-Perea, Perea Dallos Margarita).

In this regard I have a following question:

Do someboby of you have a positive experience with tree crops (woody crops) to get successful aseptic culture from the meristem? Our efforts in this area was not successful, that is why I'm asking...

I usually try to use fungicides via sterilization step, low concentrations and long time washing especially for tree crops. Moreover, I try to use the systemic fungicide treatment for the mother plants during one or two months before the in vitro cultivation.

Dear Mahboubeh Davoudi Pahnekolayi,

thank you for your response,

could you please clarify what kind of fungicides do you use for pretreatment of explants? How long to immerce and what concentratons ?

Dr Lidia Samarina,

You can also go through similar discussion on following link-

https://www.researchgate.net/post/How-to-control-fungal-infection-in-plant-tissue-culture

It might be useful to you

All the best

Use shoot tips as explant and treat explants with systemic fungicide before inoculation.

Lidia Samarina

Dear Lidia,

I usually use Carbendazim. it is better and less dangerous than others. about 0.1 to 0.2 g/l for 15 to 20 minutes is enough depending on the explant type and contamination rate.

I recommende to search with kind of fungus you have in your plants.

After a good desinfection of your shoots and under laminar flow cabinet you take

stereo-micropcope (definfected) and then you choose a meritem, transfere it to the media.

As you know meristem tissue doesn´t have vascular tisuue and not presence of mycelium, spores and others structures. So meristem tissue is free of pathogens.

Good succesess on your research.

Sterilize the explants with aqueous solution of Bavistin (systemic fungicide) prior to inoculation to avoid fungal contamination.

Many thanks to all for your valuable answers

Refer to my recommendation on September 3/2021

Many a times when we give up with other procedures as highlighted above, we use mild solution 1% or so of betadiene (povidioe) easily available as ointments at pharmacies everywhere and at homes a must have for personal cuts etc.. we found it a robust antifungal and at the same time mild to plants for a brief a day or two of cotreatments and later x3 times washing under room temp with sterile water and subculturing into suitable media. Hope it helps ur plants as did for many of ours.

Is the work of Gaurav Mudgil published?

https://www.youtube.com/watch?v=TORRxwbz7aY

I recommend spraying your mother plants by systemic fungicides and also washing your explants prior to disinfestation by a fungicide.