How to Micropropagate.

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Copyrights © Reserved Markus Axelsson, 2005.

Glass cans

You may find containers suitable for culturing plants anywhere. Jam jars and baby-food jars are excellent. It's important that the container has a certain height; allowing the plant to grow. Light has to be able to penetrate the glass, choose a clear and transparent jar. Keep in mind that the jars may have to be crushed in order to release the plants with care.

A jar with a thin aluminum lid is superb! Clean the jars and instruments that you'll use just like you normally do the dishes. Remove lables etc. from the jars.

Medium

The plant needs many nutrients and they all have to be available through the medium.

A medium called P668 or P6668 (similar to Orchimax) is a good standard-medium for culturing of most tropical epiphytes (orchids). Doesn't contain hormones (growth regulators). Use this as a medium to replant explants that are developing or to put developed protocorms on. (Protocorms that have developed may need replates).

The so called MS-medium (n.b. with micronutrients and vitamins) is suitable for many different plants, but to half the regular concentration. Doesn't generally contain growth regluators.

To succeed with propagations from explants it's often a requirement that hormones (growth regulators) are included in the medium. Such substances may be difficult finding. There is however a medium already containing hormones in suitable concentrations: P793.

It's easy to make an online order and get a well-formulated medium via mail. A simple medium can however be made from simple household ingrediences (well, I should claim that the hormones are 'household' chemicals, they must be ordered from some kind of chemical retailer).

Instructions

The fertilizer and all the dry ingrediences except the agar are measured to the right amounts and added to a 1 litre measuring-container. The banana is mashed or mixed and added to it as well. Water is filled up to the 400mL point (that is 4dl). It will not be 4 dl of water since the banana and the other substances have some volume. The mix is stirred around to a smooth mixture and transferred to a heat-tolerant container (2 litre volume or so). More water is to be added. This water can be used to rinse out all the residues that are left in the first container so that no substances are left out. 600 mL (6 dl) of water is added. The total volume is thus one litre (1L).

The mix is heated and the hormones are added. Agar is also added and is stirred down. Try not to make the medium skim too much. When the agar is in solution (sould not 'look grainy') the medium is divided up into smaller portions.

Setting the culture medium

The cleaned jars are filled up with medium to a couple of centimetres from the bottom and up. The jars may 'wander' as filled with the hot solution.

The jars are sealed with cottonplugs and aluminum foil if no metal lid is available. An extra aluminum foil covering a lid is never wrong. Don't put the lid on too tightly! The pressure may increase to magnitudes far too high for the container to handle.

Sterilizing

There are several ways of sterilising your vulture vessels with medium. A good and fast method is to pressure cook the jars. Let the pressure cooker rise to full pressure and turn the cooker off. An ordinary oven may otherwise be used. At 130-150°C the containers are "baked" for up to 2-3 hours. Tighter lids for the latter may decrease the water loss from the medium.

Also sterilize water and instruments. A jar or heat tolerant bottle is filled to ½ or ¾ of the total volume with pure water and is pressure-cooked or baked. Instruments such as tweezers and short steel sticks may be wrapped into aluminum foil or placed in a stainless steel container.

Cooling

Media containing banana and activated carbon may stratify when cooling (heavier particles sediment onto the bottom of the bottle). Sometimes the containers may have to be swirveled around a bit to homogenize the medium. When the medium is closing to temperatures less than 50°C one should let the bottles rest (agar-solutions solidify at about 40°C). The vessels can be placed in a cold water-bath.

Chose a plant - Inoculate

Some plants are a lot easier to micropropagate than others. The African Violet is a nice plant to start with. The deal is to try whatever that feels right. In the African Violet case, use leaves. Cut the leaf in half by the length. Place the leaf so that it cuts down a bit into the medium with the cut downwards. A smaller piece of a leaf may be inoculated onto the medium, resting flat with one side down completely on the medium and with the edges in contact witht the medium.

The best parts to start with when propagating orchids are the stem nodes. A centimetre or so is left on both sides of the meristem (node). The small brackt covering the node is removed and the explant (well, the stem-part-with-node) is cleaned from any plant parts that have been torn up or so. The idé is to make the explant more easily sterilized. After sterilization, the stem part is inoculated so that the node is just in level with the surface of the medium, or slightly above.

Sterilizing

Before an inoculation (see above) the explants have to be sterilized. If the parts aren't sterile or if a microbe would somehow get in to the medium, it's likely that the culture would be destoryed.

Hydrogen peroxide (2-3%) can be used as it is (can probably be bought at the chemist's). There is no need to rinse explants with sterile water after a hydrogen peroxide treatment.

Common bleach is easy to get hold of. It's often sold as chlorox or some similar name. The substance of bleach is called sodium hypochlorite and occurres with a concentration of about 5% in bleach. You will need a solution of 0.5-1%, which means that the bleach will have to be dilutet. 1 part bleach to 5 parts of water is probably well enough.

First wash the explants carefully with water and liquid washing agent (the same thing used for doing the dishes). Don't rinse the explants and don't dry them. The washing agent will make the sterilization more efficient.

Sterilize for ca. 15 minutes with the bleach-solution or ca. 30 minutes with hydrogen peroxide.

Methods and Sterile procedures

There are microbes almost everywhere in our surroundings. That means that it's important to work with extremely clean things in very clean areas and to see to it that anything that isn't sterilized is kept away from the medium. One has to work with a special method to minimize the risk of contamination. E.g. on can work inside a tilted aquarium that has been sterilized on the inside or one can work in a steady stream of hot steam, above a container of boiling water.

... read more about this in sterile methods.

Growing in vitro

After a successful preparation of the medium, sterilization of the medium and the plant parts, and inoculation, the jars are marked with information such as the date of when inoculated and what species that are cultured.

The cultures are placed in artificial ligh. (Preferably special 'grow-lights'.)