Several years ago when I was planning my nursery a friend was finishing her Ph.D. in plant science. At that time I was wondering how to establish a large collection of stock plants without having to mortgage my house. She suggested that all we had to do was use the techniques of tissue culture to micropropagate one of each of the desired varieties to obtain a huge number of plants within months. She had my attention!

So I began to research micropropagation…

Ever had that feeling that something was just too good to be true? Well it certainly was in this case!

I learnt much about micropropagation and how it's possible to produce thousands of plants from a single plant in very short periods of time. But I also learned that it was not a technique being used to propagate peonies with any success! So, my nursery was established the old fashioned way by buying stock plants from reputable peony people and waiting years while they grew. However my interest in micropropagation remained.

This article is intended to introduce the reader to the concept of micropropagation which some gardeners will be surprised to know, is widely used today to produce many of the plants we grow in our gardens. (See end of article for list)

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Micropropagation is a vegetative method of multiplying plants. There are many other methods of vegetative propagation such as dividing or taking cuttings. For example, if you want more 'Sarah Bernhardt' peonies, you dig up the one you have and divide it into a few pieces. If you want to propagate an African violet, you take a leaf cutting and induce it to produce roots. Et voila more identical plants!

To micropropagate a plant, the propagator starts with a tiny part of the plant (called an explant) and places it in an aseptic environment with an array of growth regulators and nutrients. If the environment is correct, the tiny piece of plant will begin to produce numerous tiny plantlets. The following picture shows African violets producing numerous shoots in a an aseptic growing environment.

African Violets Growing in a Micropropagtion System

These tiny plantlets are removed, divided and placed back into the multiplication environment for further multiplication.

Once enough plantlets have been obtained, the environment is changed to promote the formation of roots on the tiny plantlets. The plantlets are then moved to the greenhouse to grow into normal plants.

The advantage of this method of propagation is the sheer number of plants that can be obtained. For example if an African violet explant can produce a 3-fold multiplication every 30 days, one single explant can potentially produce over 175,000 plantlets in a year!

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Looking a little more deeply into the process…

Micropropagation begins with the concept of totipotency. Totipotency being the ability of a plant cell to regenerate an entire plant when subjected to the right conditions i.e. a single plant cell contains all the genetic material necessary to build a whole plant.

Believe it or not, scientists as far back as the beginning of the 20th century were thinking of ways in which this principal could be demonstrated and to what uses it could be put. In the 1960's George Morel in France applied the plant tissue culture knowledge of the day to orchids and the first commercial micropropagation activities had begun. He was attempting to use tissue culture techniques to eliminate virus from orchids.

During his work on virus elimination he discovered that the tiny orchid tips were actually multiplying in his test tubes! He had discovered a way to obtain many orchids from a tiny piece of orchid tissue. He had actually micropropagated orchids!

Since George Morel's discoveries in the 1960's, work has been done on many plants to discover the right conditions needed to induce tiny pieces of plants to grow and multiply in test tubes.

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The best way to describe the micropropagation process is as a five-step process.

In the first step, or Stage 0, the plant material to be propagated is prepared. Healthy plants at the right stage of growth need to be obtained.

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In Stage 1 the explant of the desired plant is established in aseptic culture. In other words, a tiny piece of plant tissue is cleaned and induced to grow in an environment free of obvious contaminants - usually a test tube, but in the picture below a petri plate.

African Violets - Stage 1

Although the concept of totipotency suggests that all plant cells have the ability under the right conditions to regenerate whole plants, in practice some parts of the plant work better than others. Dormant or mature plant tissue is more difficult to work with than rapidly growing juvenile tissue. In the above picture the explant material is the centre vein of an African Violet leaf.

The carefully selected plant material is surface sterilized with such treatments as washing with antibacterial soap, soaking in a bleach or iodine solution. Hairy tissues are harder to clean than smooth tissues and underground plant parts are often more difficult to clean than above ground parts. This cleaning of the plant material however is of vital importance. If the initial material is not cleaned properly it is possible to move pathogens into the growing environment along with the plant material. The picture below shows cultures contaminated with a fungal pathogen just 2 days after establishment.

Contaminated Culture

Once in the nutrient rich growing environment, the pathogens can rapidly overcome the tiny explant. Once the explant is surface sterilized it's placed in the growing container, usually a test tube, on a carefully prepared medium appropriate for the particular species of plant.

All of this manipulation is done using sterile technique (sterilized tools, in a special clean hooded area etc.…)

The growth medium is a critical component of the micropropagation system. It must contain all the nutrients appropriate for the plant, along with the right array of growth regulators and all in the right concentrations!

A growth regulator is another name for plant hormone. Hormones in plants, as in people, are required to stimulate cells or specific tissues into action.

It can be a very daunting task to develop a medium for a specific species of plants. The plants nutritional requirements at all stages of growth have to be know. The effects of growth regulators also need to be understood as well as how externally applied regulators will react with those already present in the specific plant.

Coming up with the right medium necessary for the explant to grow can be very much a process of trial and error. Knowledge of the requirements of other plants grown in culture and a detailed knowledge of the specific plant's growth requirements in its normal growing environment e.g. the garden, can help determine a starting point.

Experiments are carried out until hopefully a medium is found that will sustain and promote the desired growth pattern.

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During Stage 2 the explant, now established in its new environment, is induced to begin to multiply. The growth regulators present in the medium stimulate the plant material to produce numerous new shoots. (In some micropropagation systems the plants produce callous material before producing the desired new shoots)

Rapidly Multiplying African Violet Cultures

At certain intervals the micropropagator will remove the rapidly growing material from the growing container, divide it and then transfer each piece to fresh medium for the process to continue. The process of division is done under sterile conditions to ensure no opportunistic pathogens enter the growing environment.

The regular division of rapidly growing plant material is what leads to the high yield mentioned above for African violet. As mentioned above, a single violet explant cut in 3 once a month and each piece returned to culture for subsequent division would potentially produce over 175,000 plantlets in a year!

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Stage 3 is the rooting stage. At this point in the process the medium is changed to promote the formation of roots rather than shoots (as was the goal in Stage 2). The concentrations of growth regulators that promote shoot formation are reduced while those known to produce roots are increased. In some cases the light and temperature exposures are also changed to improve rooting performance.

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Once roots are formed on the plantlets they enter Stage 4 or the ex vitro establishment phase. During Stage 4 the plantlets are transferred from their aseptic growing conditions to the greenhouse for further growing on.

This adjustment can sometimes be very difficult for the plantlets. It can be likened to the hardening off process that gardeners undertake before planting out seedlings. Rather than move delicate seedlings from the greenhouse directly to the garden, a good gardener will get the seedlings used to the outdoor growing conditions slowly by exposing them very gradually to their new environment. It is much the same with micropropagated plants.

In a test tube the relative humidity is very high and the plants are provided with carbohydrates directly in the form of sucrose so the leaves have no need to protect against excessive water loss and the roots have not had to work very hard either. Once placed in the greenhouse however all this changes and the plants need time to adjust gradually to their new conditions.

Once the transition has been made, micropropagated plants grow vigorously and can often out perform traditionally propagated plants because they are disease free and for a time are still under the influence of their finely tuned growing medium.

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Conclusion

I believe most gardeners would be surprised to realize just how many of the plants around us are actually the product of an efficient micropropagation system. The following are just a very few plants that are routinely produced in vitro.

• Lilies
• Gerbera
• Hosta
• Clematis
• African Violets
• Ficus
• Boston Fern
• Carnations
• Gardenia
• Dieffenbachia
• Kalanchoe
• Rose
• Orchids
• Strawberry
• Potato

Peony will perhaps one day appear on the list also as researchers in several locations around the world are searching for the elusive system to micropropagate peony.

The opportunities brought about by tissue culture or micropropagation for the peony world are fascinating.

• Micropropagation would allow the results of selective breeding programs to be commercially available much more rapidly and probably at a more reasonable cost.
• The techniques of embryo rescue could be used to germinate in-vitro seeds that were not previously viable
• Species peonies could be more widely available without a negative impact on wild populations
• The techniques of ovule or microspore culture could lead to crosses being made that are difficult or impossible today.

In the mean time however there is still much to be said about the traditional methods of growing and propagating peonies which have served the nurseryman and gardener alike for hundreds of years.

For anyone interested in learning more about tissue culture and micropropagation, I recommend the highly readable book "Plants From Test Tubes - An Introduction to Micropropagation" by Lydiane Kyte and John Kleyn.

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