The beginning of the large-scale application of micropropagation in the horticultural industry dates from the early 1970s. Since then, it has become indispensable in propagation of many valuable floricultural crops. It is probably the most intensively growing branch of horticultural production with a hundred new firms starting to operate around the world each year.
Micropropagation is popular because of its high efficiency, the good phytosanitary status of progeny plants and possibilities to control their quality. However, this last advantage can be achieved only with a sound knowledge of physiological reactions of plant tissues to the factors by which they are manipulated.
Micropropagation, as any other horticultural technology, enables a producer to control the most important environmental factors, ie. temperature, light, media and air composition, etc. Being able to manipulate with equipment settings doesn't necessarily mean one knows how to control the growth and development of plants tissues.
Knowledge of organogenesis and developmental phenomena of many economically important species is still scarce. It is worth remembering that commercial micropropagation is in its initial stage compared with traditional horticultural technologies which have been conducted for hundreds of years.
The lack of knowledge concerning plant tissue reactions to artificial in vitro conditions is the basic reason for most of the problems encountered by horticulturists with products of micropropagation. Taking into account their impact on production results, these problems can be classified into two groups as:
- causing deviations from the phenotype (quality) standard,
- changing production schedules.
Phenotype aberrations
The changes in morphology of particular organs are often observed within plants propagated in vitro. Aberrations in leaf, stem, flower shape as well as the arrangement of leaf and/or flower on a stem have been observed.
Probably, the most common abnormality is prolific branching of progeny plants. It can be advantageous in the case of foliage plants such as Spathiphyllum, Syngonium, Dieffenbachia, and others but it can diminish the quality of cut flowers, ie. in gerbera production.
The changes of leaf and flower colours can be observed within micropropagation progeny. They usually occur during the propagation of variegated plant forms or cultivars which were obtained as the result of mutations.
In most situations, the abnormalities in plant morphology last only a few weeks after in vitro culture but sometimes they can be observed during the whole life of a plant.
Impact on production scheduling
Plants grown in vitro must be acclimatised to greenhouse conditions after planting out. This is much more complicated than the rooting of softwood cuttings taken from greenhouse grown stock because in vitro plants are far more delicate.
Some plants are very difficult to acclimatise but even with easier ones this procedure delays the start of normal greenhouse production. Acclimatisation delay can last from two-three days to two-three weeks.
It has been found that some plants (gerbera, chrysanthemum and others) obtained by cuttings in vitro begin to flower a few weeks later than plants propagated by cuttings in a greenhouse. Therefore, growing crops which are programmed to bloom in a particular week and using micropropagated plants one must take into account possibility of later flowering.
The reasons
The artificial conditions created for micropropagation are the reasons for most changes in progeny plants. These conditions are:
- artificial light of low intensity (500-5000 lux),
- high relative air humidity (approx. 95-100 %),
- unusual composition of substrate (sugars, growth regulators, etc.),
- air composition (50 ppm of carbon dioxide two hours after light is on, 1500 ppm - at night).
These conditions are necessary to enhance certain organogenetic processes but, on the other hand, are the causes of some physiological disturbances. The most important of them are:
- vitrification,
- heterotrophy,
- chimera segregation,
- rejuvenalisation,
- disturbances in morphogenesis.
Vitrification is still the ill-defined phenomenon which is widely observed in production and scientific laboratories. The most common characteristic of vitrified plants (or organs) are changes in colour to a deeper green, "glassiness" of leaves which are usually of abnormal morphology and anatomy. Vitrified plants contain much more water that normal ones and are devoid of a wax layer on leaves.
Vitrification is the main reason for problems in the acclimatisation of in vitro produced plants. At present, high air humidity, low matrix potential of media, low light intensity are blamed for the occurrence of this phenomenon.
Heterotrophy and mixothrophy are forced by the addition of sugar to culture media. Depending on light intensity, temperature and carbon dioxide concentration in flask air, plants grown in vitro can be totally dependent on an external source of carbohydrates or partly covering their needs by limited photosynthesis.
Plants grown in vitro must switch to autotrophy after planting out. This is one if the reasons for the delay in the acclimatisation to greenhouse conditions. Low concentration of carbon dioxide in closed tissue culture vessels is the main factor limiting photosynthesis in vitro.
Many valuable cultivars of important horticultural crops (carnations, chrysanthemums, apples) originated by spontaneous or induced mutations. Such plants are usually composed of tissues of different genotypes with the genotype of the most external stem cell layers dominating internal ones (periclinal chimeras) and deciding on the phonotype of a plant.
Chimera segregation occurs when new stem meristems are of adventitious nature and originate from the deeper stem tissue layers or from a callus tissue. This can take place when cytokinins in too strong concentrations are applied or when a callus stage is included in micropropagation method.
Reversion of plants to a juvenile stage can cause a delay in flowering, changes in leaf morphology and enhancement of vegetative growth. The physiology and biochemistry of this phenomenon is still not clear, however, the role of cytokinins, as the inducing factor, is evident.
Different disturbances in organ morphogenesis leading to excessive branching, fasciation of shoots, changes in leaf and petiole shape are usually the effects of the overdosing of chemicals added to media. Special care must be taken not to overdose growth regulators such as cytokinins, auxins, etc.
In general, the excess of cytokinin (within the limits of physiological concentrations) cause abnormalities in shoot development and block root formation; the excess of auxins can also inhibit shoot growth and cause deformation of developing roots.
As mentioned before, the reasons for most of the problems encountered with micropropagated plants are physical and chemical conditions of in vitro environment. Recent efforts to eliminate or diminish the negative influence of in vitro propagation head in the direction of high technology. The definition of high technology was presented by Dr Kozai during the symposium "High Technology in Protected Cultivation" in May 1988 in Hamamatsu, Japan.
Some remedies
The introduction of high technology in micropropagation means we must limit the uniformisation of media and cutting procedures and adapt them to the needs of a particular genotype or stage of propagation.
One can object and state that such a way of technology handling drastically increases the need for sound knowledge of plant physiological reactions and complexity of management operations. However, high technology offers computers to help in the management of information flow within the micropropagation firm and external data bases.
In the last few years, some efforts have been undertaken to eliminate the causes of micropropagation drawbacks. The experiments have been heading in the direction to lower humidity of in vitro environment and to enhance autotrophy of in vitro grown plants.
Debergh, with co-workers, elaborated a bottom-cooling culture system where the temperature of a shelf is 4-5°C lower than the temperature of air in a growth chamber. In such conditions the condensation of water from the flask's atmosphere occurs and the relative humidity of culture flasks is lowered by 10-15%.
Many research centers and commercial firms try to develop the culture systems where the concentration of carbon dioxide and oxygen in direct plant tissue environment can be manipulated. All these efforts are heading towards the development of the hydroponic-like culture methods where the concentration of chemicals in nutrient solution and the composition of air are adjusted automatically to the needs of a particular genotype and stage of tissue development.
However, the practical application of all these conceptual and technical developments will not be possible without the sound knowledge of physiology of photosynthesis, morphogenesis and growth of the most important horticultural plants. The technical base is there - what we need is to define the rules of its use for the benefits of propagators and growers.