from micropropagation to microponics

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From Micropropagation to "Microponics"

by Maciej Hempel

Originally published in: Practical Hydroponics International, 1993, November/December, p. 21-23

The idea of growing plant tissues on artificial media in controlled conditions had its origin at the

beginning of this century. The German physiologist Haberlandt, searching for a new investigation

method in the physiology of plant cells, did not expect that seventy years later his concept would be

transformed into a multimillion dollar, high-tech industry.

Haberlandt did not succeed in the practical application of his idea, but twenty years later his

successors developed it into a technique useful in investigations of plant development. The characterof studies dictated the use of small tissue fragments as experimental objects, to have full control

over their living processes. The small size of explants (the excised plant portion) forced the

introduction of culture sterility to protect them from invasions of microorganisms inhabiting plant

surfaces and present in air.

The first real successes in keeping small fragments of plant tissues alive in vitro for a prolonged time,

came with the discovery of auxins in the late nineteen thirties. The addition of indoleacetic acid

(IAA) to tissue culture media enabled cultivation of callus, roots and, later, shoots for arbitrarilylong time.

In the fifties, new plant hormones such as cytokinins and gibberellins were discovered. Now,

researches were in almost full control of tissue growth and development in sterile environment.

Knowledge of plant development reached the level which permitted commercial applications of

tissue culture. It started with freeing plants from viruses. This could be achieved by isolation of very

small shoot apices (meristems) and growing them on sterile media which provided for their

transformation into small plants. These plants could then be taken out of sterile culture vessels and

established in standard (unsterile) horticultural environment (see Peculiarities of Tissue Culture

Environment).

Dahlias, carnations, chrysanthemums and cymbidiums were the first plants freed from virusesthrough meristem culture. The last three of them were of big commercial potential and attracted the

attention of researchers during the next ten years. Methods used for "virus-freeing" of carnations

and chrysanthemums were straightforward. The transformation of shoot tips into plants suitable for

planting in horticultural substrates, could be achieved after one or two subcultures with the use of

one medium formulae.

The development of cymbidium in tissue culture was more complicated. Small shoot tips, when

placed on media, produced globular structures called protocorms. Protocorms could be easily

stimulated to produce new "baby" protocorms by mechanical injuries (incisions, divisions,

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scratching). This way the stock of protocorms grown in vitro could be easily increased from one to

a few million during one year of culture.

The ability to control this phenomenon gave a big stimulus to entrepreneurs in plant propagation.

The possibility of producing a few million plants from one healthy stock plant in one year seemed

incredible. Producers of planting material saw big money in the application of this propagation

method and this stimulated fast development of commercial in vitro propagation methods in late

sixties and early seventies. To lower costs of this labour-intensive production, specialisedequipment and instruments were developed. Laminar air flow cabinets providing bacteria-free and

fungi-free work environment were introduced in tissue culture production in the early seventies. At

the same time, the term "micropropagation", denoting vegetative propagation (cloning) in a sterile

environment, was coined (see Short Glossary of Micropropagation).

The seventies were a decade of boom in horticultural micropropagation. It was facilitated not only

by technical improvements, but also by the work of many researchers. The most important from a

micropropagation point of view, were investigations into the role of growth regulators in shoot

apical dominance and shoot branching. Stimulation of shoot branching is the core of the most

reliable micropropagation methods of today.

From among hundreds of researches active in the field at that time, Prof. Toshio Murashige and his

students from the University of California distinguished themselves developing practical andtheoretical foundations for today's micropropagation. They outlined the method, dividing it into four

stages (see Stages of Micropropagation). They also developed commercial micropropagationmethods of many important ornamental plants such as Gerbera, Nephrolepis, Syngonium and many

others. Media developed by them in early seventies are still the most commonly used in manycommercial laboratories.

The application of labour intensive, high-tech and, therefore, expensive micropropagation has been

economically justified only in the case of ornamental plants. This situation has not changed duringthe last twenty years. The need for sterility of cultures dictates the use of specialised and expensive

equipment. Small dimensions of objects and the need for their careful handling during subcultures onfresh media, lower labour efficiency. Cost of labour constitutes 60-70% of total micropropagationcosts. The increasing costs of labour during eighties caused the closure of many micropropagation

laboratories in developed countries. In USA, only 150-180 laboratories were active at the end ofthe last decade, in comparison with 500 in operation at the beginning of the eighties.

Reduction of micropropagation costs can be achieved by its mechanisation and automation. It is a

difficult task because the structure of plant objects grown in vitro is very complicated. Only a fewplants, like eucalypt or potato produce simple elongated shoots with clearly visible internodes and

leaves. Most ornamental plants branch intensively during culture producing thickets of entangledshoots of different sizes. Efficient automatic division of such structures is impossible at present levels

of knowledge in 3-D computerised vision systems. Additionally, difficulties in sterilising complicatedrobots and accessory equipment must be added.

But do we really need sterility for fast cloning of plants in fully controlled environment?

As previously mentioned, sterility of cultures was introduced to prevent bacteria and fungi from

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growing on media containing sugar. Media have been supplemented with sugar because cultivated

tissues were so small that they could not feed themselves. It is certainly still true at the beginning ofthe micropropagation process when very small tissue fragments are isolated from stock plants.

However in most micropropagation methods applied nowadays, these small explants develop laterinto complete plants with shoots, leaves and roots and are potentially capable of photosynthesis.

They do not photosynthesise efficiently because of insufficient supply of carbon dioxide and too lowlight levels in container environment (see Peculiarities of Tissue Culture Environment).

What would happen if we enhance gas exchange between the culture flasks atmosphere

and growth chamber environment and provide more light to sustain photosynthesis?

Many research centres have been trying to answer this question during the last eight years. Theirfindings are astonishing. It has been proven that many plants can be grown and forced to branch in

small containers on sugar-free media if concentration of carbon dioxide and light levels are highenough to enable photosynthesis. In many experiments, growth was even better than on media withsugar. Plants produced on sugar-free media tend to acclimatise and grow faster in horticultural

environment than plants from media containing sugar.

These findings stimulated the development of culture containers and container closures whichfacilitate control of flasks atmosphere. Many different solutions have been proposed. The simplest

are gas-permeable membranes incorporated into container closures; the most complicated aresystems were composition of container air is constantly monitored and adjusted when necessary.

The bigger the containers, the easier is the control of their environment. Dr Kozai and his studentsfrom Chiba University developed the culture system were containers are a hundred times bigger

than traditional tissue culture vessels. He grew plants on liquid media in a greenhouse where lightand carbon dioxide levels were fully controlled.

Is it micropropagation or sterile hydroponics? Or may be "microponics" - because of the small

dimensions of cultivated plants. Probably, we do not need sterility of cultures any more, but onlyprotection from plant pathogens. This could lead to more open propagation systems, speeding upthe introduction of the latest achievements in robotics and the reduction of propagation costs.

Micropropagation would then be accessible for many important vegetable, agricultural and forestrycrops.

Peculiarities of Tissue Culture Environment

Tissue culture is conducted in containers which are sealed to prevent colonisation of media and

tissues by microorganisms. Relative air humidity inside containers reaches almost 100%. The light

level used in growth chambers to trigger developmental processes is low (approx. 40 mol/m/s) and,therefore, not sufficient to sustain photosynthesis. The closure of culture containers restricts gas

exchange between their interiors and the growth chamber environment. Carbon dioxide content can

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drop down to 100 ppm two hours after lights are switched on. It is much below the level required

for normal photosynthesis.

These changes in the "normal" plant environment have a profound influence on the development of

aerial parts of plants. The surface and internal structure of leaves and stems are altered and

resemble that of aquatic plants. High air humidity changes the structure of cuticle, wax deposits,

stomata and mesophyll cells. Sugar is added to culture media as a source of energy becauseconcentration of carbon dioxide and light levels are too low for efficient photosynthesis.

Specific tissue culture conditions have a big impact on growth and development of plants after they

leave culture containers. During the first week after transfer to a horticultural environment, plants"learn" how to photosynthesise, open and close stomata and uptake water and nutrients through a

root system. This transition period between tissue culture and further cultivation is called

acclimatisation .

Short Glossary of Micropropagation

In vitro - (Latin: "in glass"); experimentation or cultivation of an organism(s) or a

portion of it in glass or plastic ware in artificial conditions.

Tissue culture - cultivation of plant parts: cells, tissues and organs, under aseptic conditionsin/on synthetic media in vitro.

Growth chamber - a chamber used for incubation of culture containers or plants under a

controlled environment.

Micropropagation - vegetative propagation by application of tissue culture; it is usually

conducted in growth chambers.

Propagule - a portion of an organism (shoot, leaf, callus, etc.) used for propagation.

Explant - the excised plant portion used to initiate a tissue culture.

Subculture - the aseptic division and transfer of a culture or portion of that culture to a

fresh nutrient medium.

Stages of Micropropagation:

Stage

I

-establishment of small fragments of stock plant(s) in tissue culture.

StageII

- multiplication of propagules; the most common method is through stimulation of branchingand subsequent division of shoot clumps on smaller explants which are then placed on a

fresh medium. Through repetition of the process, number of initial propagules can increase



a million times in one year.

Stage

III

- preparation of propagules for transfer to normal growing conditions through rooting or

elongation of shoots.

Stage

IV

- establishment of Stage II or III propagules in normal growing conditions - usually in soil or

potting mix in a greenhouse.

from micropropagation to microponics

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