Dear Mesnoua,

          Microshoots with extensive root system are washed thoroughly in tap water to eliminate the residues of the agar and transferred to plastic pots in a mixture of sand and peat in 1: 2 (v/v). The plantlets are acclimatized for six weeks under tissue culture conditions. The pots are covered by translucent plastic bags to maintain high humidity and prevent the dissection of the newly transferred plantlets. The plastic bags are pored (one pore/ five days for five weeks) to decrease the humidity and acclimatize the plants to external atmosphere gradually. After six weeks, the plastic bags are removed completely and the plants are transferred into black plastic bags (35 x 25 cm) contained a mixture of sand and peat in 1:1 (v/v) and allowed to grow under open conditions for further two months until reach height of about 30-40 cm. Thereafter, when regenerated plants become strong and having woody stem, they are transplanted to the field and show normal growth and phenotype.

Note:

Before using the sand, it must be washed several times until the washing water becomes clear, then it is dried, and after that it is used.

Best regards.

In my opinion before going for Physical Hardening you go for Chemical hardening by gradually simplifying lowering conc of media constituents for one or two transfers. Then go for Physical hardening to get good success of survival.

I transfer each of my transgenic plants (ex. cotton) from medium to a soil pot (soil is wet). Then cover the plants with a cover (ex. a Magenta box) to maintain high humidity and leave a very small 'crack' for air-exchange. Every one to two days, I will increase the size of crack a bit. Continue to do so, until the plant establishes itself in the soil, then I will totally remove the cover. This process can take more than 10 days. When I did so, I avoided sunlight directly hit on those plants. That may increase the temperature inside the cover. For doing so, I have reached more than 95% of acclimatization survival rate (of transgenic cotton).

It depends on the plant species. For tissue-cultured tobacco, it is much easier in terms of acclimatization.

Please see similar discussion on RG: https://www.researchgate.net/post/How_can_in_vitro_wilting_be_prevented