5.11.25 How to grow orchid seeds with B1 fungus

Part 3: Sow seeds and add fungus

If you have any questions about Parts 1 to 3 please feel free to e-mail me (see below).

Firstly, a word about safety. Follow the described methods entirely at your own risk. However there should not be anything very dangerous. Bleach is harmful so follow the safety instructions on the bottle. I am not aware of the B1 fungus being harmful and I have been using it for nearly 20 years with no problems. It is, however, strongly advisable to avoid personal exposure to B1 and any or all other microbes growing on Petri dishes. Contaminating microbes on nutrient media certainly could be harmful to health.

Having prepared Petri dishes of Basic Oats Medium in Part 2, it is now time to add orchid seeds and B1 fungus. Use seeds of Common Spotted orchid (Dactylorhiza fuchsii) to start and practice with as they are readily available and grow well with B1.

Traditionally the seeds are surface-sterilised of adhering microbes before sowing. This is done with dilute bleach for just long enough to sterilise externally but without killing the seeds. Bleach is then removed by a water rinse before sowing to stop it inhibiting growth of the fungus.

Make a 10% dilution of Domestos bleach with tap water. Put a small “pinch” of dry seeds in an Eppendorf tube then add approx. 1mL of dilute bleach. Shake to mix thoroughly. Incubate for at least 15 minutes, mixing periodically. Do all subsequent steps in the cabinet.

Now slowly decant off the liquid leaving most of the seeds behind, stuck to the tube wall. Don’t worry about losing a few! Add approx. 1mL of the sterilised tap water that was cooked in the pressure cooker in Part 2. Mix thoroughly and slowly decant off the liquid again. Finally add another approx. 1mL of sterile water. Mix to resuspend the seeds. Give the tube a final shake and ASAP throw the contents briskly onto the centre of the medium in a Petri dish.

Fluid and seeds coalesce in the centre. Disperse them around evenly by gentle side-to-side shaking and tilting. There will be excess water left on the surface which encourages contaminating bacteria. Tilt the dish steeply and hold for a while to make it run off to one edge. Remove as much as possible with a pastette.

There is plenty of seed left stuck in the tube so add another 1mL of sterile water and repeat the sowing process with a second Petri. This way you get two sowings from one tube. Inevitably some seed is wasted during decanting and sowing, but never mind! 

Finally add a piece of agar from your B1 colony to each Petri. Using sterilised scalpel and spatula cut a cube with sides approx 1cm or a little more. Transfer this to the seed Petri and deposit it, fungus face down, on the agar. It can be in the centre or at the edge, wherever you prefer. Label the Petri with the date and all relevant information. I use a marker pen. This is easily wiped off later with methylated spirit when you want to re-use the Petri.

Petris must be sealed to prevent dehydration. You can use parafilm or sticky tape etc but I like to seal them in small individual press-lock plastic bags. This stops the lid from coming off and prevents moisture loss. Store in the dark at room temperature. A drawer in a cupboard is ideal. Observe what is happening at regular intervals. The fungus should grow out and cover the plate in about 1 to 2 weeks. In about 6 weeks you should be able to see small protocorms with the naked eye.

There is an easier way to sow the seeds. I like to think of this as “Tony’s Way” although no doubt other people use it too! I add the B1 fungus cube to the Petri first, and this can be incubated for a few days to make sure you can see fungus growing out successfully into the medium. When B1 has just started to grow out dry seeds are added in its path.

The surface sterilisation step is completely omitted. Simply brush the seeds out carefully from the fold in a piece of paper so that they fall all over. Use a soft water-colour brush. You only need roughly 100 to 200 seeds per Petri.

The simplicity, speed and efficiency of dry sowing onto a Petri where B1 growth is already confirmed make it very attractive. The disadvantage is that seeds sometimes carry heavy contamination with surface microbes. They grow rapidly and spoil the medium. In this case it is necessary to use the surface sterilisation method. That doesn’t happen often though, and you usually get away with it!

There is another way of adding the fungus too. When you progress to the stage of having spare orchid protocorms that are too small to bother growing on into plants you can use them for fungus transfer.

Move one or two of these with sterilised forceps to the sowing Petri and the B1 will grow out well. This is a quick, simple, and clean technique which is usually very successful.

 In future Blog posts I will probably address some fine tuning of the seed sowing process and then describe how to “grow on” protocorms when they have reached a few millimetres in size.

Anthony.heys@sky.com