Proliferation of nodal segment excellent on dkw without contamination but after 3 week it died... I try subcultivating every week on new dkw but not good? Maybe after proliferation need some diferent media and hormon concentration... Any help?
Hi
You can use DKW medium with 0.5-1.5 mg/l BA to multiplicate walnut shoot tips.
Your cultures were died because the browning. The walnut tissues contain high concentration of phenols. You must to add antioxidants like activated charcoal, PVP (Poly vinyl pyrrolidone) or 100 mg/L ascorbic acid + 150 mg/L citric acid. Thank you and good luck.
Dear Dejan, in vitro establishment is the harsher and unpredictable stage of walnut micropropagation. Since 2006, I have established several tens of different walnut genotypes. For each established genotype I have failed with 3 of them. The most important factors are genotype, explant source and the expertise of technician. According to the picture, shoot seems vigorous, however, I miss the formation of basal calli. It's an important factor that determine that an introduction is reacting well to tissue culture conditions, at least in my case.
Dear @Ricardo, what would happed if he excise the shoot from basal tissue that seems darker, and transfer the shoot into a fresh basal medium or direct root induction medium. However, in this case rooting must be undertaken... It seems the shoots in Magenta boxes are bothered with high humidity, why don't you keep the boxes away from the light after removing the excess water in it. I'd also insistently suggest you to follow Ricardo's walnut micropropagation paper.
Buhara, that's a nice proposition: shoot is big enough to growths by itself. Condensation might be also a consequence of a reduced exchange, proper of tight closed vessels. In any case, it must be avoided.
Mahamadreza, remember that phenolics aren't the only substances that are released to culture medium. How plants react them exactly is not completely clear. Phenolics can be detected "easily" because of oxidation, other substances not. Difussion into agar is slower than in liquid media, therefore all the released substances tend to accumulate around explant when gelled media are used. When an explant (in my experience and in my case) feels "nice" under tissue culture condition reacts, first, forming a basal callus and, later, growing vigourously. Always from my point of view, until you don't get this state of "confortability", frequent subcultures should be done. Here in RG (https://www.researchgate.net/project/Second-generation-of-planted-hardwood-forests-in-the-EU-WOODnat) I have uploaded some pictures on the establishment of several genotypes of walnut, a Pesian walnut among them. May be these can help.
Dear Ricardo, thank you for your advice ...all you have specified we conduct
how much is in our capabilities... thanks for the link to project i will follow that it is very interesting to me.
Ricardo I have a question for you: protocol in the project is from the previous works (improving with Fe-EDDHA) or you are modify it?
Mohamadreza we'll try with Fe-EDDHA (accordig to Ricardo paper) finaly we got a chemical Fe-EDDHA (Duchefa)!!
Thank you very much dear colleagues !
Since 2015 I don't have introduce new modifications to protocol. However, nowadays we're trying to adapt it to commercial production, so it's highly probably that some changes must be done. I also use FeEDDHA from Duchefa. Thanks for your interest in our project.
