Preparation of media
To make 1lt of media. … Using a container with a volume of at least 1.5 or 2 litres first measure out 900ml of the water you choose to use (reverse osmosis , de-ionised or distilled water, avoid any other type.) Add the powdered media mixture of your choice, – you may also need other ingredients such as agar, sucrose and activated charcoal if it not already present -, rinse out the original media mix container with a little extra water, add to the 900ml then top up to 1lt. .. stirring occasionally, gently heat to boiling either in a saucepan or in a microwave, ensuring that the agar is fully dissolved before pouring media into your vessels.
When it is ready, carefully dispense a small volume of media into each of your vessels, to a depth of around 1cm, to keep the charcoal in suspension stir the mixture after every three of four vessels filled. Close containers as appropriate but be sure to leave screw tops unscrewed by half a turn and to place a small tab of aluminium foil under the lid of plastic vessels. Wrap the tops of the vessels with aluminium foil and load vessels into your pressure cooker … include a small piece of autoclave indicator tape if you have some. Securely close the lid then quickly raise the temperature of the cooker until it reaches 15psi ( a loud whistling or hissing should be heard), from this point set a timer for 15minutes. More time than that may damage the media and less will not ensure that media is sterilised so try and time carefully. As soon as the 15minutes has elapsed, remove the cooker from the heat and allow to cool until the cooker lid can easily be opened.
Take the cooker/autoclave to your prepared workspace, remove the lid and place the containers of media into the prepared workspace to cool, use gloves and dip your hands into the workspace sterilant before they enter the sterile workspace. Once the media has cooled and set, tightly close the lids of the containers, they can then be removed from the work space and stored in a cool dark place, retain the aluminium foil on the lids, it keeps them clean. Try and leave the prepared media for a week or so prior to use, discard any that become contaminated.
Note:- you may not need to use all of the media at one time, it is possible to freeze for later use, particularly where you add your own agar… just freeze without agar.
Sowing seed onto sterile media.
Prior to sowing the seed onto your sterile media, it is strongly recommended that you prepare the seed in advance. As has already been noted, orchid seed is extremely small yet it still has in place barriers of a chemical or physical nature which, in the wild, serves to prevent them from germinating at the wrong time or place.
Both freshly collected and previously dried orchid seed are likely to be water repellent to some degree and will almost certainly be contaminated with spores of a fungal or bacterial nature. These pre-sowing treatments are designed to overcome these barriers.
The flowing guidelines are for sowing mature dehisced seed, either fresh or previously dried and not for the ‘green-pod’ method. It will not matter which equipment you choose to use, pipettes, syringes etc. the method will be the same and can be adjusted to suit.
Pre-sowing treatment.
Prepare first-soak water by pouring around half of a pint of de-ionised water into a suitable container. Add about one-quarter teaspoon of sucrose … ordinary table sugar … and a drop or two of plain washing-up liquid or, if you have it, Tween 20.
Prepare vials of seed.
Add a tiny quantity of seed to a small capacity vial of about 4-5ml or a small capacity syringe (even a tiny amount will represent several hundred orchid seeds).Now add some of the previously prepared first-soak water and replace the lid of the vial and lightly agitate, the orchid seed will very likely float, put to one side for 24-36hrs.
The thoughts behind this procedure are twofold, first the sucrose is thought to encourage any spores to develop and thus be vulnerable to the killed by the sterilization procedure to follow and second that the wetting agent (washing-up liquid or Tween-20) will overcome the water repellence of the seedcoat and allow the embryo to fully imbibe.
After the 24-36hr period, remove the soak water, if the seed has all sunk then it is ready for sterilisation and sowing. If the seed has not all sunk then it may be beneficial to repeat the soak treatment but omitting the sucrose, the aim is to have all of the seed sink to the bottom of the vial or syringe … some species take longer than others since some have a greater degree of repellence. Empty seed will be unlikely to sink and it would be advisable to remove it if you can.
Once seed has sunk to the bottom of the vial or syringe remove all of the soak water, rinse with fresh, clean de-ionised water and remove, leaving the seed in a few drops of clean water, it is now ready to sterilise and sow onto the media.