Acclimatizing tissue culture plantlets from the vessel to the greenhouse

Stage IV

The move that undoes good work

Acclimatization is the last stage of micropropagation, and for many labs it is the expensive one. A shoot can multiply cleanly for months, root well on a good stage-III medium, and still collapse within days of leaving the vessel. A grapevine study in the journal Vitis describes micropropagated plantlets as generally susceptible to rapid desiccation at lower humidity, and calls the acclimatization procedure costly in labor and equipment.

The reason is not mysterious. Inside a sealed vessel the plantlet lives in near-saturated air, at low light, with sugar in the medium and almost nothing to fight off. The greenhouse offers drier air, brighter light, a nonsterile substrate and a living population of fungi and bacteria. Every one of those changes arrives at once unless the lab spreads them out.

This piece covers why plantlets fail ex vitro, what can be done before they leave the vessel, how to handle them at transfer, and how to step the environment down. It ends with the part most labs skip, which is counting survival in a way that tells you something about the lines and recipes that produced the plants.

The plantlet

Why plantlets fail ex vitro

Start with the leaf surface. A cuticle and its layer of epicuticular wax are what keep water inside a leaf. In vitro leaves tend to carry far less of it. The grapevine study estimated lower wax deposition on micropropagated plantlets than on acclimatized and field-grown plants of the same clone, and a 2016 study of the orchid Cattleya xanthina in Biologia Plantarum found epicuticular wax deposition increased during acclimatization.

Then the stomata. Leaves formed in saturated air often have stomata that do not close normally when the plant starts losing water. In the grapevine work, a large majority of stomata on detached in vitro leaves were still open 3 h after exposure to 63 % RH. A plantlet in that state cannot slow its own water loss, so it wilts in conditions an established plant would ignore.

Third, the plantlet has been living partly on the sugar in its medium. A 2020 review of ex vitro establishment in fruit crops in the International Journal of Current Microbiology and Applied Sciences describes this heterotrophic habit as a root cause of weak transplants, and notes that heterotrophic conditions and long dark periods leave plantlets with poor photosynthetic capacity. Out of the jar, the sugar is gone and the plant has to feed itself with leaves that were not built for it.

Finally, the roots. The same review notes that roots formed in shallow medium are often short and have a less developed vascular system than roots formed in soil. A root system that cannot keep up with transpiration is the fastest way to lose a plantlet, which is why the first days after transfer are mostly about keeping water loss low while new roots form.

Stage III

Start hardening before the lid comes off

Some of the work can be done while the plantlet is still in the vessel. The 2020 review cites earlier work showing plantlets can be partly hardened in vitro by lowering humidity and raising light before they are taken off the medium. Ventilated closures are one way labs lower humidity inside a vessel. In the grapevine study, vessels with covered vent holes held relative humidity at 94 %, against 100 % in vessels with intact lids, during the last stage of culture, and the reduced-humidity vessels cut transpiration from the leaves.

This is also where stage-III recipe choices show up later. Rooting medium, culture period, closure type and growth-room light all shape the plantlet that arrives at the bench. A lab that changes any of them should expect acclimatization results to move too, and should be able to tell which change it was.

Plantlets should be well rooted before transfer, but practice on timing varies by species and protocol. A 1995 study in Pesquisa Agropecuária Brasileira that acclimatized four species across several rooting ages and substrates found survival depended mainly on the species. That is a useful reminder that one transfer schedule will not suit every line in a mixed lab.

Transfer day

Washing, substrate and hygiene

Take plantlets out of the vessel gently and keep them from drying while they wait. Work in small groups so no tray of bare-rooted plantlets sits under the bench lights.

Wash the medium off the roots. The 2020 review describes plantlets being separated completely from the medium, with thorough washing of roots to remove it. Agar left in the root zone still carries the sugar and nutrients the plantlet was living on, and growers generally remove it so it is not sitting in a nonsterile mix where fungi and bacteria can use it. Clean water and patience do more than force; torn roots set the plant back.

For the substrate, the University of Florida tissue culture glossary describes transplanting well-rooted plantlets into a standard pasteurized rooting or soil mix in small pots or cells. Labs use a range of mixes, and the right one depends on the crop. What matters most is that it drains, holds air around the roots, and is clean.

Hygiene carries over from the lab, even though the greenhouse is no longer aseptic. Clean trays, covers and tools, fresh substrate, and a bench kept apart from older stock plants all reduce the load a weak plantlet has to face. None of this guarantees a clean batch; it simply removes some of the easy ways to lose one.

Environment

Step humidity down and light up, slowly

Right after transfer, the plantlets need protection from drying out. The University of Florida glossary describes a shaded, high-humidity tent or mist or fog, and notes that several days may be required for new functional roots to form. The 2020 review describes humidity kept near about 95 % under plastic sheets or inverted glass jars in the first phase, then moisture reduced gradually before plants move on to further hardening under partial shade.

The principle behind every schedule is the same. Lower humidity in steps, not in one move, and raise light the same way. Venting a cover a little more each day, or moving trays from a misted bench to a drier one, gives the new leaves time to build cuticle and the stomata time to start working. The Cattleya study concluded that a gradual acclimatization gave that species the best adaptation to its new environment.

Watch the plants rather than the calendar. Leaves that droop or curl after a cover is opened are telling you the step was too large; close it up again and try a smaller one. New leaves that expand normally in the drier air are the clearest sign a batch is ready for the next step. Exact humidity levels, light levels and durations vary by species and protocol, so treat any published figure as a starting point for your own records.

Verify this

Before a batch leaves the lab

Confirm each tray is labeled with its lot, line and stage-III recipe, and note the date it came off the medium.

Count the plantlets going in, so the survival figure later has a real denominator.

Records

Count survival per lot, not per season

Most labs know roughly how acclimatization is going. Fewer can say which lines, recipes or transfer dates lose the most plants, because survival is counted for the greenhouse as a whole, or not written down at all. A loss that is not tied to a lot cannot be traced back to anything.

A useful record is short. For each lot, note the line or clone, the stage-III medium and culture period, the closure type, the date of transfer, the substrate and cover or mist used, and the number of plantlets moved. Then count survivors at the same checkpoints every time, for example at the end of the covered phase and again when plants leave the hardening bench. Note the likely cause of losses where you can see one, such as wilting, rot at the base or damping off.

Over a few cycles, that record shows patterns a season total hides. One line may acclimatize poorly on every recipe. One rooting medium may produce plantlets that do well on the bench, or fail soon after transfer. A run of losses may line up with a particular week, room or substrate delivery. Keeping these records per batch and lot, linked to the recipes and lines that produced them, is what lab software like xPlant Pro is for.

Acclimatization will never be the stage where nothing goes wrong. It can be the stage where you know why.