A white structure is forming after 5-6 days of explant inoculation (without contamination), subsequently the material get contaminated. I tried 1 mg BAP, 2 mg BAP, 5mg BAP+0.5mg NAA etc., and the results were same. What may be the reason? will you suggest related publications?
might be due to an efficient sterilization techniques (physical and chemical, I prefer using different type autoclaves and chemical solution to sterilization
Dear Subin Kunnath it's a classical behaviour of walnut's explants when it comes from actively growing materials. During the second week after in vitro introduction, petiole usually fall down along with the formation of this kind of friable calli. In the case of picture, explant looks phenolized, almost dead: under this circunstance it's not rare the flourishing of endophytics. What species are you working with?
Subin Kunnath This is due to the formation of the abscission layer under natural conditions and the petiole with the leaf used to fall down after maturity. But under in vitro conditions due to the presence of more cytokinins in the medium the cells involved in the abscission process get activated and start cell division (make callus) and the left portion of petiole falls down. This phenomenon is quite normal in tissue culture woody species. Regarding getting contamination during this process is maybe due to problems in the sterilization process.
Ricardo Julian Licea-Moreno Hydnocarpus sp. , An endangered tree from Western Ghats, India.
How to overcome this problem sir? any related publications?
Dear Subin. I think there is formation of tiny somatic embryos inside the circle in the Fig I attached. The Camera resolution is so low that we can't see this development well. May be this treatment is from high BAP? The white amorphous appearance, in some plant species is formation of somatic embryos or embryogenic callus inside it. The petiole breaks and fall due to lack of auxin, these area is the Abscission zone. I don't see (I'm not sure) development of endophytic fungus. Following such browning color of the stem, the internal part could remain alive and I've seen such phenomenon in my work. If there is endophytic fungus you will see mass of greyish powder, which is formation of spores. Don't discard it and wait what will happen.
Subin Kunnath I don't know the species; however, since it's from India I must to assume that it's not a decidous tree, as walnut. Anyway, it doesn't matter. Bonga, during 80's, stated that a critical key to succeed with tissue culture of trees is to use juvenile explants: the higher the degree of juvenility, the higher the success probability. There're several ways to obtain juvenile explant sources. From the selection of the youngest branches to the grafting, passing through methods to force trees (or part of trees) to produce young branches. However, it doesn't assure the success, so an important part is your patience. Probably you'll need to introduce a lot of plant material before obtaining viable explants. Wish you success!!!!
It appears to me that you have here two things, one is the formation of Callus and the 2nd is the contamination. You will need to go over your sterilization procedure first to eliminate your contamination and then to work your propagation procedure if you go directly from your start tissue into proliferation or through callus.
It seems your sterilization method is not appropriate and plant is exudating phenolics and producing callus like tissues for its petiole.
To me it looks like callus, it's not a problem per-say, just an unwanted side effect. I used to see something equivalent with incubation of cut poplar twigs. The callus didn't disrupt the actual experiment (flush of dormant floral buds).
What are you hoping to do with your explants? Grow roots? You could try removing and sub-culturing the callus itself then inducing root formation with auxin (followed by shoot formation and elongation).
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