10.01.26 How to grow orchid seeds with B1 fungus

Part 4: Growing-on protocorms

Let’s say your Petri dish from Part 3 has been incubating in the dark for 8 weeks or more now. With luck, many orchid seeds have germinated and are developing into protocorms. Germination involves imbibement of water and swelling of the spherical seed embryo as its cells divide and grow. It bursts out of the surrounding testa and becomes a small white structure with conspicuous root hairs (rhizoids). One end of the protocorm is usually tapered and often brownish in colour. This is thought to be where the fungus is present internally. The opposite end is white, more rounded and swollen, and bears the first shoot just beginning to poke out.

 The rhizoids may be important for absorbing water and minerals, and promoting entry of fungal hyphae. It is certainly possible for seeds to germinate into tiny protocorms without the aid of the fungus. They run out of nutrients at this point if they are not supplied by an appropriate fungus in the soil. A fungal culture or an artificial nutrient medium can sustain them in vitro. In our case we have little protocorms “plugged in” to the B1 fungus, drawing carbohydrates and other nutrients that they require. On a successful plate there may be 50 or many more crowded little protocorms competing with each other and they must be “thinned out”.

Just as a gardener “pots on” seedlings to keep them growing well the next step for the protocorms is to move the largest, healthiest ones on to fresh medium. Those about 3mm long or more are big enough to survive the move and thrive. Smaller ones may well be too damaged if you try to move them. By doing this we thin the initial sowing Petri dish and allow smaller protocorms to bulk up. Those moved on get a greater share of the nutrients than they previously had and grow faster. Good for everyone!

We can re-visit the sowing plate after a few weeks and move on a second crop. There is a limit to how many it is worth moving on from the first plate, however. The weaker ones are not worth the effort, so maybe harvest only the best 20 or so?

Make a number of Petri dishes of fresh Basic Oats medium in just the same way as described in Part 2 for seed sowing. Use a fine-tipped pair of forceps that has been sterilised, and work inside the sterile cabinet. Gently pick up selected protocorms and transfer them to the fresh medium. I generally put no more than 5 or 6 on one Petri dish, evenly spaced out.

The protocorms are surprisingly hard - like little nuts - but try to be gentle and not mangle the root hairs too much. I usually grasp them just behind the shoot bud. Also make sure they have stuck to the new surface before whisking the forceps away, as they can remain stuck to the latter sometimes!

Label the new Petri dish and seal it in your preferred way. Incubate again in the dark at room temperature for two weeks to allow the fungus to grow out and the protocorms to settle in and fatten up somewhat. At this point it is convenient to give them a Winter, in case they need it to trigger development later on. The first shoot starts to grow out now but is not quite ready to turn green, so it can be kept in the dark. Place the Petri in a fridge at 4 deg. C for 6 weeks. There will still be some growth in the protocorms during this time.

After 6 weeks move the Petri back to room temperature but this time place it anywhere in moderate room lighting. Avoid very bright light such as a south facing window ledge. The shoot now elongates, turns upwards and goes green. Allow 2 weeks or so to re-acclimatise to room temperature growth.

The shoots are now bumping up against the Petri lid so it is time to transfer the protocorms to a jam or honey jar of fresh medium. This is also beneficial to keep them growing strongly. I use 100mL of sterilised B.O.M. medium per jar made up as usual, but you could use more. It can be autoclaved directly in the jar as the glass is tough enough. Have the jar lid on, but only loosely. This prevents volume loss from the jar but avoids any pressure effects that might break the glass.

After the medium has cooled and set I add up to 6 protocorms per jar, around the edge. Do this with forceps in the sterile cabinet, as above.

The jars are placed in moderate light and temperature. While the protocorms are still small there is no need for ventilation with external air. This will come later; see below. The lids can thus be fixed on tightly. Over the next 2 or 3 months the protocorms will slowly grow into small plants with up to 2 or 3 green leaves and the first proper roots developing.

This program of transfers is fine for most orchid seeds that germinate with B1, eg all the Dactylorhiza species, Herminium monorchis, Goodyera repens etc. It needs to be modified, though, for species that grow their shoot very rapidly upwards such as Anacamptis morio and Anacamptis laxiflora. The first transfer from the sowing plate has to be to a jar, not a Petri, to give enough headroom.

The jar can’t then be put in the fridge because it has to be in the light as the shoots go green quickly. Just put the jar in moderate light in a fairly cold place during winter. Don’t let it freeze, though! There is one less transfer between mediums this way but it doesn’t matter. Strong little plantlets form within a couple of months.

Once you reach the jar stage there isn’t much to do for a while except admire your plantlets. It may be a good idea, though, to make “breathers” in the lids after about a month to allow exchange of gases with the outside air. I always do, just to be safe.

You can change or modify lids without having to use sterile conditions as soon as the fungus is established. I discard the plastic or metal lid and stretch a double layer of polythene bag over the top secured by a rubber band. Make 2 or 3 small holes in the middle with a pin and cover these with a double layer of microporous tape. It seems to work!

There are obviously many other ways of doing this. More recently I am using kitchen aluminium foil as a cap instead of the polythene bag. It can be used from the start when autoclaving the medium, instead of the plastic lid.

That’s it now until about March, April, or May when we have to start thinking about what to do with the plantlets! Please do e-mail me if you have questions.

Anthony.heys@sky.com