Walnut (Juglans regia L. family Juglandaceae), is a temperate nut crop. As one of the most important nut crops, the annual world production is 1491,567 metric tonnes from an

area around 646,748 ha. After about 5–6 weeks intact plants with roots, stems and

leaves are formed on all the responding media. In the microplants the roots are  more robust and developed than the leaves. Yes,  composition of agar media changes for few day for in vitro hardening before moving them to greenhouse. Agar  media is a polysaccharide, due to temperature effect it dries and become hard or it become stif due to sol-gel interactions in humid conditions .Always use fresh media for culture never, use stored media as its upper surface get harder.

At the stage of moving to greenhouse just environmental condition is very important. If you keep container on the pots for 2 weeks and gradually remove them, your planlets can tolerate in vivo condition. 

You shall transfer the rooted plantlets to small earthen pots containing ‘Soilrite’ (Keltech Energies Ltd. Bangalore, India) and cover it with transparent polythene bags to maintain 90–99 % relative humidity. Kept in 25 ± 2 0C temperature and 20 h photoperiod for 30 days. Thereafter, the hardened plants are transplanted into

earthen tubs containing soil and maintained inside the poly-green house (30 ± 2 0C temperature, 14-16 h photoperiod and relative humidity of 60–65 %) for another 14 days, thereafter the acclimatized plants will remove from poly-green house

and transfer to field condition under full sunlight.

Best of luck.

You also see the following papers...

Haque and Ghosh Botanical Studies 2013, 54:46

http://www.as-botanicalstudies.com/content/54/1/46

OR

Haque SM, Ghosh B. 2014. Applied Biochemistry and Biotechnology 172:4013-4024.

DOI 10.1007/s12010-014-0817-2

OR

Haque SM, Ghosh B. 2013. National Academy Science Letters 36:551-562.

DOI 10.1007/s40009-013-0161-z

In the in vitro hardening step you can add paclobutrazol 0.5-5mg/L for 2-3 weeks and then go for ex vitro hardening. Paclobutrazol induces more chlorophyll content, thickness of leaf, epidermis, cuticle; reduces transpiration rate, thereby plants become very conservative in utilizing resources and becomes hardy also. Please let me know the results if you follow this steps.

Thanks. I will try paclobutrazol

Parm

On the basis of the results obtained in my research, the best procedure for rooting of Juglans regiaL. microshoots consisted of two phases: in vitro root induction and ex vitro root development.  For root induction the most efficient method was utilisation of DKW medium (half strength macronutrients) solidified with 2.4 gl-1 Phytagel containing 30 gl-1 sucrose, 4.0 mgl-1 IBA and incubated in dark conditions.  For root development, shoots were transferred from root induction medium, washed with tap water, planted in moistened Jiffy-7 pots, sprayed with 1.0 gl-1 Benomyl solution and placed in propagator with 90-95% relative humidity in light conditions.

In This procedure, root development was synchronised with shoot elongation, new leaf formation and acclimatisation. This procedure which consisted of a nine days in vitro root induction period, and six weeks for root development and acclimatisation is a short and economical method for rooting of Juglans regiaL. shoots.  By transferring the plantlets in Jiffy-7 pots to larger pots, the roots of plantlets were not damaged and survival of planlets was high.

Thanks. We will test 9 day dark induction. Seems pretty long time to keep plants under dark conditions. I wonder what kind of survival rate you get in the greenhouse. I notice that J. regia makes roots but roots are too strong (contrary to J.hindsii x J.regia Paradox rootstocks).    

Thanks, You can decrease the induction time to 7 days. The survival rate was about 90% for well developed roots and shoot.

How do your plants look in vitro?  If they are vitrified, you will likely have poor results no matter what you do.  If this is the case, you need to look at your protocol to see if you can prevent the problem.  If your plants look healthy in vitro, the previous suggestions are all good.