Dear Badhepuri Mahesh Kumar

Reports suggest that sugar remobilization and ‘N’ uptake are the major causes of defoliation in vitro.

The non-availability or deficiency of C (glucose, fructose, sucrose, fructans), and N (amino acids, soluble proteins) in the nutrient medium leads to de-foliation.

Increased level of hormones and ethylene accumulation can also cause defoliation, use cotton plugs and avoid vent tape to seal your culture jars.

Defoliation can also be due to the Exudate produced by the plants/ explants/ shoots

Sometimes gelling agents also plays significant role in defoliation in vitro, use of phytagel could reduce the defoliation at certain level.

1. Increase the level of Sucrose and nitrogen source in the medium

2. Increase the supply of vitamins particularly Vit-C

3. Increase the light intensity as well as duration of culture illumination and reduce the temperature of culture incubation.

4. Understanding physiology and nutritional requirement of the species before culturing in vitro can be highly useful in developing a successful protocols.

The following links may be useful

doi: 10.1093/jxb/ery211

https://agrilifecdn.tamu.edu/briske/files/2014/03/Briske-Richards-SRM-CHAPTER95.pdf

Dear Badhepuri Mahesh Kumar as M. Manokari has mentioned, sugar and N might cause in vitro defoliation. However, some other factors must also be considered. Microbial contaminants; the kind of vessel; volume of culture medium; explants inoculated per vessel; lenght of subculture; plants species; nutritive formulation; agar brand; kind and hormone concentration; among other factors, alone or combined can provoque in vitro defoliation. I hope it helps you.

I have also observed defoliation in Hemidesmus indicus cultures especially when growing In presence of cytokinins. Addition of low auxin concentration helped me.

In addition with above answers, you can go through earlier discussion on same topic which might give you other ideas

https://www.researchgate.net/post/Defoliation-of-in-vitro-cultures

All the best.

Thank you Ashutosh Pathak Sir

There are good suggestions on the link which you have shared...!

And We are also working with Hemidesmus cultures in our laboratory...! BY making little modifications our Hemidesmus cultures are growing good

Thank You Ricardo Julian Licea-Moreno sir... I make a note of your suggestions

Thank you M. Manokari Maam

I will try to implement your suggestions

Following the constructive views of dear Ricardo Julian Licea-Moreno, I think this phenomenon can be attributed to the reduced in ability to absorb nutrients from the culture medium and medium contamination.

Thank you Reza Ghahremani

Yes Reza Ghahremani, your view is correct. Defoliation is due to the inefficiency of explants to absorb available nutrients in the medium and sometimes particular deficiency micro and macro elements to fulfill the in vitro response of the selected explant/ plant.

One of the reasons could be the deposition of macro and micro elements in the culture medium.

Firstvof all, What is your plant species?

Thank you Ilknur Eskimez

I will try to make modifications in the medium

Buhara Yucesan the plant name is Codiaeum variegatum (L.) Blume

Dear Badhepuri Mahesh Kumar leaf defoliation can also occur when the temperature drops and different lighting modes

Sometimes the use of silver nanoparticles may help to stop leaf senescence. Even the accumulation of CO2 and ethylene gases also promotes defoliation.

Thank you Mahipal Shekhawat Sir

Thank You Serik Jantassov

Even, increased concentrations of Iron in the medium could also work to prevent defoliation of cultures in vitro.

Thank yoy Mahipal Shekhawat Sir

I will try this increased iron concept

As Mahipal Shekhawat have mentioned, improving iron nutrition also might help correcting yellowish, and defoliation. In my experience I've began to use FeEDDHA instead FeEDTA, and besides other abnormalities, chlorosis and defoliation were removed. You can see here in my RG profile a couple of papers where these results are mentioned :

1. Improved walnut mass micropropagation through the combined use of phloroglucinol and FeEDDHA

2. Unraveling Factors Affecting Micropropagation of Four Persian Walnut Varieties

Dear Ricardo Julian Licea-Moreno Sir

I am also hearing this FeEDDHA thing more frequently nowadays...

Going a little deeper into FeEDDHA, if you wouldn't mind may I know the concentration and procedure of how were you using FeEDDHA in PTC?

We are really interested to use this in our experiments...!

Thank you, Sir

Dear Badhepuri Mahesh Kumar with walnut I've used 119mg/L. The procedure is quite simple: just weight the quantity you need, and disolve it in water, that's all. I've used reactive grade FeEDDHA; however, my collaborators in Kazakhstan have used a commercial brand: with few experiments you can adjust the quantity you'll need.

Ricardo Julian Licea-Moreno Have you tried filtered caps. This will help to reduce ethylene.

Dear Kourosh Vahdati , yes, I've tried. In fact, I've been using Microbox vessels from SacO2 with green filters.

1. Try to use a culture flask with a cotton plug for better aeration.

2. Increase Fe and Boron contents in the medium.

3. Subculture regularly.

Thank you Ricardo Julian Licea-Moreno Sir

Defoliation, usually occurs as a result of in vitro ethylene production. You can use ethylene inhibitors like silver nitrate or modify medium composition based on the experiment, defolition usually observed in tree species, woody shrubs and Climbers, with MS medium with high concentration of BAP, I would like to recommend WPM and Kinetin for such plants. For more information about role of ethylene in plant tissue culture

you can check: https://doi.org/10.1007/BF00024072

Thank You Mafatlal M. Kher Sir

for sharing valuable experiences

I will try that

One more thing you can try is liquid culture medium as it will increase the aeration as suggested by Mahipal Shekhawat sir.

Thank you Ashutosh Pathak sir And Thank you Mafatlal M. Kher Sir

Dear Dr. Kumar,

I think this symptom hasn't occurred to all kinds of plant species. Vessel cap with holes might help to reduce ethylene concentration accumulated inside the culture vessel. How about the pH of of culture medium? Have you ever tried to adjust pH, before autoclaving, equal or higher than 6?

Best regards,

Quynh T. Nguyen

Respected Quynh Thi Nguyen Madam, You are absolutely correct, yes all the plants won't show the same kind of response but the gathering of previous experiences helps us troubleshoot things. and pH was adjusted to 5.80 before Autoclaving.

The new thing I got from your answer is aeration to the cultures

I will immediately apply this to the cultures and I will see the response. Thank You for sharing your experiences

You should use anti-ethylene compounds such as AgNO3 or use filtered caps.

Badhepuri Mahesh Kumar for which plant species do you have defoliation problem in vitro? Is it woody? If so, during lignification process under in vitro conditions may result in high ethylene accumulation, so you must degas it somehow. Most of the answer given above is highly recommended, but your question needs more clarification especially for the target crop.