It's important what kind of plant growth regulators you use for medium supplementation, and also the jars should not be closed too tight (read about ethylene).

I hope that it will work for you.

Ethylene is the main factor but it could depend on the agar amount

Subculture timing is also very important. If the time between two culture passages is two long, media will be deprived of nutrients and senescence/ ethylene production might occur . If this was not the problem, try to revise medium composition.

Actually it depends on many factores ;the medium, concentration of carbon you are using and the GRH ratio ,Also I agree with the previuos comments about the features of the cotainer and the gas exchanges.

Good knowledge

Hi Onur,

In our callus cultures, we had a problem with something similar, in that it would grow for a while, then suddenly yellow and then turn brown and then die, all within a month. This is different I realize than defoliation, but maybe our experience can help yours. We changed the media and added more vitamins. For ours, it helped to add vitamin C, take out one thing- yeast extract- that we had been using, and add more sugar as it's carbon source. We also had been using Phytagar. Then we had a good suggestion to try Phytagel. It is clearer than Phytagar, and really seems to help keep the nutrient and hormone 'flow' going into the cultures for the required 4 weeks before subculture. I hope this helps, even in ideas of changing the media to a different recipe.

The above comments about ethylene are correct. Additionally, take into account the concentration of auxins. A high auxin concentration induces ethylene production.

This is common problem in gooseberry. I worked on micropropagation of Indian gooseberry (Emblica officinallis L.). we also face same problem, try to reduce phytohormone (auxins) concentration. and avoid ethylene accumulation in culture by using vent tape to seal your petri dishes (or providing ventilation using culture bottles having cotton plug) or related provisions.

A way to deal with this by transfer of the explants onto an new medium remove alle the leaves. These leaves are sources of ACC the precusor of ethyleen. By removing the leaves you prevent high ethylene build up in the jars.

Succes, Maurice

I am also suggest that you can try to increase Nitrogen, Calcium, Boron and/or Copper concentration in the medium

Ethylene is the main factor in this scene. Improve gas exchange in culture flasks or add sodium thiosulfate or AVG on culture medium

What about silver nitrate for etylene inhibition?

Ya, it due to accumulation of ethylene or ABA. all suggestions above are acceptable

Hi Onur,

Many factors can cause the effect that you described. What kind of homone and doses are you using? What time elapsed from beginning of culture to the moment in which defoliation appearing? What culture medium are you using? Jessy have right, sometimes vitamins could help but not all the time if, for example, a mineral deficiency is causing defoliation; on the other hand when Phytagel is used deficiency of Ca and/or Mg can rise up. Are you checked the pH of culture media at the end of subculture? Microbiological contamination can also provoke defoliation in cultures.

Changing the media such added more vitamins,and reduced the concebtration of growth hormone,especially 2,4-D.

Adding Silver nitrate to the medium will help as it is ethylene inhibitor.

Hi Onur,

We had a similar experience with rose microshoots and this was overcome by adding STS. However, STS had also unwanted effects and therefore we changed to adding a small lid with Power Pellets on top of the medium. Power Pellets are grains coated with KMnO4 that oxidize ethylene to H2O and CO2. There are several similar products like Power Pellets commercially available.

But looking to your description, there are arguments that it is not ethylene, especially because you observed the problem after transferring to jars which have a klarger headspace (presuming that the number of shoots is the sam.)

Good luck, Geert-Jan

I aggree with explanation of Prof. G-J De Klerk. I had similar problem with the cultures of Bulgarian rose (Rosa damascena). Generally, this type of problem has been observed with the plants with high scandary metabolites.

To some extend you can solve the problem by taking care of ethylene accumulation. Our experience is that it does not solve the problem completely.

The alternate could be to increase the light intensity as well as duration of culture illumination. Further you may also try reducing the temperature of culture incubation. Hope these will solve the problem

Try not to use aluminium foil to cover your culture vessels but use sterile cotton bungs instead- promotes aeration - but you can cover the cotton bungs with aluminium foil when autoclaving-

From a physiological point of view leave abscission is due to decreasing auxin content in leaves due to low photosynthesis level (as an index for plant that leaves has become a consumer from a producer), or high stem auxin level by use of auxin in medium.

Both conditions lead to lower ratios of leave/stem Auxin content which triggers the and abscission process including ethylene production.

Thus reduction of auxin in medium and/or increase of light energy could help.

Reduction of sugar in medium makes plantlets more dependent on photosynthesis by leaves and can reduce the abscission.

In general any change in favor of autotrophism would be beneficial for retaining the leaves.

This conclusion is based on physiological concepts and obviously needs to be validated in practice.

The ideas of Ebrahim Hadavi was helpful.

I have used Drl Ebrahim Hadavi approach with micropropagation of soybean varieties. The length of light exposure could be helpful and transfer your plantlets more often to avoid drying out of the media. Good luck.

Defoliation can also be due to the Exudate produced by plant. Reduce 2,4D and IBA use as they enhance the production of senescence factors when used in high concentrations. Avoid any of the hydrocarbons with Ethyl group during surface sterilization as they have effect of creating an abscission layer as soon as there is subculture. Proper maintenance of Photo period must also be taken care of.

ALL THE BEST for your Work.

Thanks for all your attentions.. Firstly i tried to solve my problem with silver nitrate. Now that plant seems healthy (before all leaves were dead and plant was weak) I will prepare my own media next time and try these suggestions..

If you are using any auxins, try to reduce the levels. I tried it with my sweet potato cultures and it worked.

I think that the problem is caused by either ethylene/ ABA. If you use ethylene or ABA inhibitors, you can get over this problem of defoliation (abscission) easily

Dear Friends 

We are interesting to develop in vitro culture of gojiberri. Can you please help with protocols and medium receipes

Dear Onur Güven

We had similar problems in Vitex species, we have overcome using meta topolin and increased concentration of ascorbic acid and increased concentrations of nitrogenous stocks in the medium.

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 the duration of culture illumination and reduce the temperature of culture incubation.

There are several causes for the effects of light intensity, temperature, and auxin secretion, which can cause leaves to fall off. Root aeration and mechanical stress due to the transfer of seedlings from Petri to pots can also cause leaf fall. Therefore, it is better to transfer the seedlings quickly and in the best conditions of humidity, light, temperature, etc.