9/17/13 From Micropropagation to Microponics - pub

file:///C:/Users/H P/Desktop/home group/BM SINGH/Monal/tissue culture technology/plant tissue culture/From Micropropagation to Microponics - pub.htm 1/5
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 character
of 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 arbitrarily
long 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 viruses
through 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,
9/17/13 From Micropropagation to Microponics - pub
file:///C:/Users/H P/Desktop/home group/BM SINGH/Monal/tissue culture technology/plant tissue culture/From Micropropagation to Microponics - pub.htm 2/5
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, specialised
equipment 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 and
theoretical foundations for today's micropropagation. They outlined the method, dividing it into four
stages (see Stages of Micropropagation). They also developed commercial micropropagation
methods 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 many
commercial 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 during
the 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 on
fresh media, lower labour efficiency. Cost of labour constitutes 60-70% of total micropropagation
costs. 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 of
the 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 few
plants, like eucalypt or potato produce simple elongated shoots with clearly visible internodes and
leaves. Most ornamental plants branch intensively during culture producing thickets of entangled
shoots 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 complicated
robots 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
9/17/13 From Micropropagation to Microponics - pub
file:///C:/Users/H P/Desktop/home group/BM SINGH/Monal/tissue culture technology/plant tissue culture/From Micropropagation to Microponics - pub.htm 3/5
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 of
the micropropagation process when very small tissue fragments are isolated from stock plants.
However in most micropropagation methods applied nowadays, these small explants develop later
into 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 low
light 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. Their
findings 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 high
enough to enable photosynthesis. In many experiments, growth was even better than on media with
sugar. 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 which
facilitate control of flasks atmosphere. Many different solutions have been proposed. The simplest
are gas-permeable membranes incorporated into container closures; the most complicated are
systems 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 students
from 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 light
and 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 only
protection from plant pathogens. This could lead to more open propagation systems, speeding up
the introduction of the latest achievements in robotics and the reduction of propagation costs.
Micropropagation would then be accessible for many important vegetable, agricultural and forestry
crops.
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
9/17/13 From Micropropagation to Microponics - pub
file:///C:/Users/H P/Desktop/home group/BM SINGH/Monal/tissue culture technology/plant tissue culture/From Micropropagation to Microponics - pub.htm 4/5
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 because
concentration 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 conditions
in/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.
Stage
II
- multiplication of propagules; the most common method is through stimulation of branching
and 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
9/17/13 From Micropropagation to Microponics - pub
file:///C:/Users/H P/Desktop/home group/BM SINGH/Monal/tissue culture technology/plant tissue culture/From Micropropagation to Microponics - pub.htm 5/5
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.