You can try to change benzyladenine for some other derivative from topolin family (hydroxy or methoxy derivatives of BAP) (Aremu et al., 2012; Malá et al., 2013; Valero-Aracama et al., 2010 and others).

Perhaps the BA is inhibiting rooting. Did your shoots elongate 1-2 inches prior to auxin induction? Rooting in plugs? what medium? Do you get rooting in vitro if you transfer to hormone-free media, lower salt? Did the microcuttings get stressed out in the fog chamber? wilting?

Rodney can be right, actually the inhibitory compound is usually major metabolite of BA, 9-glucoside. That is why I suggested to use different cytokinin with different metabolism

You could try a subculture without growth regulators before putting the shoots in rooting medium, you can also try NAA for rooting (Montalbán et al. 2011). These papers could be helpful: Fernández et al. (2000) and Ermel et al. (2000)

Rodney, I did elongate 1-2 inches prior to auxin induction. I'm rooting in elle pots. the microcuttings were actually growing. i saw new leaves emerge and older leaves grew larger.

Thanks for the advices. I will try them out!

What medium in the ellepot? Coir/perl/peat? Are you doing several lines of walnut? Is this just the most recalcitrant line?

I believe it's miracle grow potting mix. I'm only working with VX211 and it's not the most recalcitrant line...

miracle grow mix might be too "hot". try fine vermiculite alone. or something like sunshine mix: peat/perlite. squeeze the water out of saturated miracle grow mix, and measure the EC. Are u sure the cuttings aren't drying out?

I'm sure the cuttings aren't drying out because i sprayed water constantly when i transferred the plants to elle pots and the elle pots were re-wetted before i put in the plants. I will try the peat/perlite mix though. Thanks a lot!

Did you plant the ellepots in the lab first? Or out in the greenhouse? How much shade over the fog bench?

I put the ellepots in the humidity dome first where it retains most of the moisture (the humidity dome mimics the fog chamber on a smaller scale). In the humidity dome, they receive the same amount of light as if they were in the lab

Light needs to be higher than in the lab. around 1500-2500fc mebbe. are your cuttings vitrified in any way? all agar, not gelrite?

You could also try applying some level of nutrient stress by using 1/2 and 1/4 DKW salts and reducing concentration of the carbon source to 15 or 10 g/l sucrose. These strategies have worked for many species.

Rodney, cuttings are all healthy, i didn't root the vitrified ones. I'm using all gelriite, no agar.

Eliud, Thanks for the advice, i will try it with reduced salts and sucrose.

Gelrite can promote vitrification. The fact that you had vitrified shoots indicates that. Is there contraindication for using agar instead of gelrite? Try comparing shoots grown on agar vs. Gelrite. Hyperhydric cultures have very abnormal anatomy, and have harder time acclimating to the greenhouse.

I'm using gelrite instead of agar because it has better clarity and i use less gelrite/liter than agar which saves money.....

Fresh cutted ones...

your shoots look a bit chlorotic (Fe?). which iron chelate are you using? edta or eddha? the necrotic leaflets: is this after being in the fog chamber? Are u decreasing humidity after 2 weeks?

Can you root an easy walnut line alongside this one? as a control? (Sunland?) I would also try an agar control, esp. after passing it 2-3 times on agar.

http://hortsci.ashspublications.org/content/39/2/324.full.pdf

Hi, I had similar problem with rooting of Aesculus hippocastanum. The problem was overcame by using metatopoline besides benzyladenine (BA). Ba metabolite BA-9-glucoside is known as inhibitor of rooting. So I recommend you to use

1/ lower concentration of cytokinine,

2/ alternative cytokinine, for example metatopoline

3/ to add active charcoal in medium before rooting to remove BA and BA-9-G residuas.

Good luck!

"However, the acclimatization of micropropagated walnut

was reported to be a difficult procedure because of rapid

desiccation of plantlets or their susceptibility to diseases

due to high humidity and difficult rooting."

In vitro propagation of walnut - A review

by K Payghamzadeh - ‎2013

www.ajol.info/index.php/ajb/article/download/92211/81665‎

I'm using EDDHA at 39.1mg/L. i do notice chlorotic leaves when i use EDDHA as iron chelate, but i also notice faster growth. When i use EDTA, I see a much slower growth rate but no chlorotic leaves. sometimes, I got necrotic leave when i put the plants on rooting induction medium and sometimes after being in the fog chamber. I will try the 15uM IBA. thanks!

Thanks Helena, I will try your recommendations!

Here's the plant on root induction medium for 3 days

I got this rate from UC Davis walnut improvement program....It's 39.1 mg/L EDDHDA in media

I hope it will improve the rooting and wish you good results!

Dear Wang

1-you need 4 weeks to rooting

2- decrease BA

3- decrease salt of DKW 1/2 , 1/4 ,3/4 

Hi. How is your walnut micropropagtion project going? Are you doing commercially? I am giving consultation to produce walnut commercially to micropropagation labs. Let me know if there is a demand to my services in your country. Attached you will find the photos of a commercial tissue culture lab and orchard that I am giving consultation.

Hello, My name's Anut

I'm research assistant

Now, I study about Dracontomelon dao I want to know about information for tissue culture it. For

callus and induce multiple shoots.

I have a question.

1. I should used medium for plant tissue culture?

2. Which hormones are used to induce the calluses and how much is the concentration?

3. Which hormones are used to induce the shoots and I want to how much is the concentration?

I hope you give an answer back to me.

Best regards

Dear respected Anut,

The images you submited show that you have explant browning in your work. So my first recommendation is to control the production phenol and I suggest adding PVP in medium culture. Due to the tissue browning, it is better to use culture medium 1/2 or 1/4 MS. Also, In my experience, the best hormone to induce callus is TDZ.