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Meristem Tissue Culture of Orchids

Tissue culture was first used to produce orchids by Dr G. Morel a French botanist in the 60's. The application of this method to the orchid plant was in fact a spin off from another process involving potatoes. The process is mainly used for mass production of a specific cultivar and for the propagation of rare, difficult to grow species. 

Tissue culture in orchids essentially involves the removal of meristem tissue which can be found in growth buds, germinating seed, nodal joints and any other part of the plant which is undergoing rapid growth changes. Once these parts of the plant are removed they are referred to as explants. The explant at this point and all following stages has to be kept absolutely sterile otherwise microorganisms which may be present will grow and destroy all the developing cells. These explants are capable of mass proliferation if given the correct growing environment, this cell mass is non specific and generally looks like tiny green balls all clumped together. The tiny green balls are in fact developing plant embryos and are called protoplasts. This cell mass as a whole is referred to as a callus. 

The callus grows in a nutrient mixture which is designed to make the cells proliferate but which prevent it forming leaves and roots, the process continues until the callus is large enough for dividing and either re-implanting in fresh medium and grown on again in to more callus (protoplasts) or it is divided and placed on a solid nutrient gel where it is encouraged to grow roots and leaves and develop in to plants.

That is a simplified summary of the process but as you would expect with a scientific method their is a lot more involved which we have explained below.

Full Process

We have broken down the process in to it's various components to describe each part in detail.

Meristems

The meristem is the central portion of a rapidly growing shoot, bud or other part of the plant where cell division is taking place. The meristem is a mass of cells which have not yet been programmed by nature to become whatever part of the plant it is from e.g. meristem taken from a flower bud would under normal circumstances eventually turn in to a flower, but as we take it before the change has occurred we can grow it on into other parts of the plant.

Explant

The material removed from the centre of a  meristem is called an explant.  This explant is a mass of cells which has no specific purpose and is capable of growing on in to any part of the plant e.g. it could develop in to leaves, roots, stems or flowers.  This material has to be sterilized by immersing it in a sodium hypochlorite solution or a 5% solution of household bleach. This sterilization is needed to remove any microorganisms such as fungal spores, bacteria and any other type of contaminant which would grow and destroy the developing cells  

It is at this point that the difficult stages begin. The rinsing and all the following stages must be conducted in an aseptic environment. In commercial laboratories they have glass cabinets which have fans built in that blow a constant stream of sterile air over the working area, these are called Laminar Flow Hoods. This stage is essential as it prevents the reintroduction of the contaminants we have just removed.

After the explant has been immersed in the sterilizing fluid for a period of approximately 7 to 10 minutes (depending on it's size), it must be rinsed using sterile de-ionized water. 

Growing Medium

Growing media formulations specifically made for orchid  tissue culture can be purchased in kit form. They are available in a wide variety of types as each orchid genera has specific requirements in terms of what nutrients and hormones are required and in what combinations. The kits purchased may or may not contain some or all of the following ingredients:

  • Macronutrients:  calcium (Ca),  magnesium (M), nitrogen (N), phosphorous (P), potassium (K) and sulphur (S). 
  • Micronutrients (trace elements): boron (B), cobalt (Co), copper (Cu), iodine (I), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn).
  • Vitamins: nicotinic acid, pyridoxine (B6), riboflavin and thiamine (B1)  
  • A food source: Usually in the form of glucose, sucrose and myo-inositol
  • Growth Regulators (phytohormones) * : Both auxins (IAA, NAA) and cytokinins ( BA). 
  • Solidifying Agent: The most commonly used is agar  

A mixture which contains all of the ingredients except the growth regulators is know as a 'base mixture' 

* Phytohormones may be added to the base mixture to stimulate the growth and development of the explant in a particular way, this is known as somatic embryogenesis.  Normal embryos formed by fusing two germ cells can grow and develop in to plants naturally but cells using tissue culture need to be induced to grow roots and shoots. In very general terms auxins are required for root initiation (but not further growth) and cytokinins for developing vegetative growth buds. Both auxins and cytokinins are required for the formation of a callus (next stage), this process of controlling the growth of root and shoots is known as Organogenesis.

Once you have mixed your growing medium according to the pack instructions it must be placed in to some kind of glass vessel, usually these glass vessels are either conical flasks or some other similar glass container such as honey jars which must be sterilized by autoclaving. A house hold pressure cooker will suffice for this. Your containers containing 1/2 inch of growing media need 'cooking' for between 10 and 14 minutes to sterilize them. If you are using screw on lids then you will need to put these on and then unscrew them half a turn, this will ensure your vessels don't break under the pressure whilst avoiding water getting in to the containers.

After the initial 'cooking' time the jars or flasks should be allowed to stand in the pressure cooker until cool enough for the lid to be removed but before it gets too cool. As soon as it is safe to remove the pressure cooker lid you should open it and immediately fully screw on the lids of the jars without removing them from the cooker. As they cool they will form a vacuum and the contents will be sterile. These jars can be made in advance and kept in your fridge until needed.

Callus

In the world of tissue culture a callus is a clump of protoplasts which have been cultured on a nutrient rich growing media (without the phytohormones) that has been kept agitated, this agitation prevents the callus from growing roots and shoots,  the callus will continue to grow until the clump of cells is either divided and then the process repeated or is divided and grown on in a different growing media.

Once the callus is large enough to be divided (depending on how many plants you need) it needs to be divided in to smaller clumps of protoplasts which can be sown on to the surface of a fresh growing media, this time containing the phytohormones. The small clumps of protoplasts can be cut from the main callus but this has to be done under totally sterile conditions, this is a highly specialized process.

Under sterile conditions the prepared protoplasts are placed in to the surface of one of your prepared jars of sterile growing media and the lid replaced. You can now place the closed jar with the explant sitting on the surface of the growing media in to a warm and light situation. Avoid direct sun as the pots will overheat.

You will know within two weeks whether or not you were successful with your sterilization as bacteria will rapidly grow and you will see this on the surface of the nutrient jelly. If you see signs of contamination remove that pot to avoid contaminating any other jars. If you were successful you should still have the tiny green protoplast clumps looking fresh on the jelly, this will eventually (6 to 12 weeks) start to grow and both roots and shoots will begin to develop.. The development varies according to the genera being produced but they can remain in the jelly for up to 12 months or until the container is full of small plants and there is no mire space for further growth.

The growing media and plants are then removed from the container and the young plants potted on in to either individual pots or communal trays. These new plants are used to high levels of humidity and will need to be weaned off this gradually by keeping them either in a specially created closed section of your greenhouse or by misting them several times a day for two to three weeks. After this acclimatizing they can be placed in your normal growing place.

This process can take up to 4 years to provide a flowering size plants, depending on genera.

Can I do it at home?

This is not really a propagation method which is suitable for doing at home, having said that, if you like to try things then you can certainly have a go using some of the kits that are available. By far the most difficult aspect of the whole process is keeping everything absolutely sterile.

A method that is commonly used with some success is to use a large wide rimmed cooking pot half filled with water, this is brought up to the boil to give off a wide plume of steam vapor. Inside this veil of steam the atmosphere is sterile and so where we mention a Laminar Flow Hood you can substitute your wide rimmed cooking pot.

The steps

Firstly prepare some jars of growing media (liquid form as this has to be agitated) and place them beside the cooking pot which should be boiling

Remove the meristem from the eye of a back bulb or from the growing tip of a shoot

After sterilizing the explant for approximately 10 minutes in a 5% solution of bleach take it over to your now boiling pan of water - while still in the sterilizing solution

Bring a bottle of de-ionized sterile water over to the pan also, this is for rinsing the explant

Pick up your dish with the explant and sterilizing solution and bring it in to the plume of steam

With your other hand rinse the sterilizing solution from the explant using your fresh sterile water - still within the steam, put down the bottle of water

Using your free hand bring the sealed jar of phytohormone free growing media in to the plume of steam, using both hands (this is tricky as you are also still holding the dish with the explant) remove the lid from the jar - still within the steam

Use a sterile instrument such as a sterilized knife to lift the explant from the water and place it in to the growing media - still in the steam

Replace the lid on the jar, tightly.

This has to be agitated (constantly moved) to prevent roots and shoots forming, hobby shops sell small electric rolling machines designed for polishing gem stones, they come in a variety of types but they should have a model suitable for this purpose.

You will notice that the most important aspect of the above method is to KEEP IT IN THE STEAM as this is your aseptic and sterile environment.

If everything goes well you should after a couple of weeks still have your explant sitting in the nutrient liquid, if bacteria or some other contaminant has got in to your jars or the explant wasn't sterile you will have mould or slime growing on the gel, remove the jars as they may contaminate your other jars.

After (6 to 12 weeks) you should have a large clump of protoplasts (embryo plants) called a callus.

Once the appropriate amount of callus has been produced (depending on your purpose) it is then divided and placed on fresh growing media (under sterile conditions) containing a higher concentration of the appropriate phytohormones (auxins and cytokinins) which will induce the non specific cells to develop roots and shoots, this is then grown on until the vessel containing the growing cells is full of tiny plants. This second stage does not need agitating as we are now encouraging the roots and shoots to grow from the protoplast. They will remain in the bottles or jars until the plants are large enough to be transplanted (20 - 52 weeks).

The growing media and plants are then removed from the container and the young plants potted on in to either individual pots or communal trays. These new plants are used to high levels of humidity and will need to be weaned off this gradually by keeping them either in a specially created closed section of your greenhouse or by misting them several times a day for two to three weeks. After this acclimatizing they can be placed in your normal growing place.

This process can take up to 4 years to provide a flowering size plants, depending on genera.

 

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