TECHNICAL FIELD The invention on the one hand relates to a micropropagation method implemented by a bioreactor and a liquid culture medium container, which are in fluid communicating connection through a connecting tube to enable the supply of a liquid culture medium. Furthermore, the invention also relates to a micropropagation apparatus for carrying out the method. The invention can be used primarily in phyto-bioreactors which are designed to run on liquid culture medium and which are mainly set up to propagate and cultivate aseptic plant parts.

BACKGROUND ART Micropropagation is a branch of horticultural (and generally plant) biotechnology introduced across the world and featuring a number of practical and scientific traits. Exceeding sales of one billion plants per year recently, the micropropagation industry has been characterised in recent years by a high manual labour intensity (with a ratio representing about 70 to 75% of the prime cost), which makes the price of propagulum substantially more expensive. In addition, as a result of the accumulating impact of a number of technical and biological problems, in vitro micropropagation is not yet competitive today with traditional clonal propagation in the case of many species. Due to these problems, the production activity of large scale micropropagation industry has been mostly transferred to the developing countries, primarily to China and India. Researchers believe unanimously that the immediate future of micropropagation industry may primarily depend on the success of technical and technological progress and secondarily on the acceleration of target-oriented biological research activities. The research aimed at this special area is characteristically targeting the development of special micropropagation bioreactors. A common feature thereof is that cultivation takes place on liquid culture medium. The inoculated explants and plants exposed to the culture medium on a larger surface grow faster and healthier than those in traditional agar-agar solidified culture media. The intention is to ensure the optimal growth of in vitro cultivated/micropropagated plants by an increasing number of new technical approaches. Reviewing the main trends and achievements of international progress of recent decades, it can be said that prior art micropropagation bioreactors make little or no use of advanced technical and technological approaches.

In vitro plant tissue cultures growing in a liquid culture media has been used for a long time. Micropropagation bioreactors which are controllable and have a periodical liquid culture medium supply have been described in US 4,669,217 and US 6,753,178 B2, in Hungarian patent application No. P 04 01841 and in JP 2000041166. The disadvantages of prior art solutions are that special pressure transmission units and compressors are required for operating the periodical liquid culture medium supply, and that they are implemented with complicated tilting mechanisms. On the one hand, prior art approaches are costly, and on the other hand they make it difficult to provide an aseptic environment. Prior art documents do not deal with ensuring an aseptic atmosphere, and do not provide for a simple change of the composition of the headspace of the bioreactor atmosphere and the liquid culture medium.

DESCRIPTION OF THE INVENTION

The object of the invention is to provide a new micropropagation/ plant tissue culture method and apparatus which overcome the deficiencies of prior art approaches. The object of the invention is to find a solution through the application of which the plants, plant tissues and cell cultures and/or inocula (the offsprings inoculated into culture medium) may be periodically exposed to the liquid culture medium in the culture vessels of bioreactors in a way that in the meantime the composition of the headspace atmosphere and the liquid culture medium can be altered directly via tubes. A further object of the invention is to provide an aseptic environment as simply and efficiently as possible.

These objects have been achieved by the inventive method described in claim 1 , and by the apparatus described in claim 6. Preferred embodiments of the invention are defined in the dependent claims. BRIEF DESCRIPTION OF DRAWINGS

Preferred embodiments of the invention are described below with reference to the accompanying drawings, where

Figs. 1A and 1B are schematic side views of a micropropagation apparatus embodying the invention in two different positions of the bioreactor and the liquid culture medium container,

Fig. 2 is a schematic side view of an apparatus comprising two liquid culture medium containers,

Fig. 3 is a schematic diagram of an examplary bioreactor shown with the cover lifted off,

Fig. 4 is a schematic side view of an embodiment which enables regulating the composition of the bioreactor headspace atmosphere, and Figs. 5 to 9 are diagrams depicting experimental results of applying the invention.

EMBODIMENTS OF THE INVENTION

The essence of recognition serving as a basis for the invention is that the cultivation chambers of the micropropagation phyto-bioreactor are gravitationally supplied with a sterile (aseptic) liquid culture medium from separate liquid culture medium chambers in a programmed way. This is preferably carried out by regulated replenishment without reinjection and/or by the in situ replacement of the consumed liquid culture medium.

In the apparatus shown by way of example in Figs. 1 A and 1 B, such a bioreactor 1 and a liquid culture medium container 2 are applied, which are in a communicating connection via a connecting tube 3a enabling liquid culture medium supply. In accordance with the invention, a further connecting tube 3b is applied for establishing a communicating connection between the headspace atmospheres of the bioreactor 1 and the liquid culture medium container 2, and the amount of the liquid culture medium in the bioreactor 1 is regulated by changing with an actuating mechanism 5 the interrelated vertical positions of the liquid culture medium container 2 and the bioreactor 1. Only the actuating mechanism 5 panels supporting the bioreactor 1 and the liquid culture medium container 2 are depicted; according to the invention, any suitable mechanism can be applied for this purpose.

Consequently, the horizontally located, preferably translucent sterile (aseptic) cultivation vessel (bioreactor) communicates with a sterile liquid culture medium container (liquid culture medium vessel) physically separated, and yet connected via flexible tubes 3a, 3b in a way that the micropropagation offsprings (e.g. the plant parts, tissues or cells) are in the cultivation vessel (bioreactor) and the liquid medium is in the culture medium container. The latter is lifted up and down along a vertical axis, to make sure that the liquid culture medium freely flows into the cultivation vessel (bioreactor), supplying the cultures with nutrients and appropriate plant growth regulators, at the same time making sure about the continuous replenishment and/or aseptic in situ replacement of the consumed culture medium using appropriate regulation. In this way, in vitro plant tissues, cells, and other sterile and/or non-sterile parts, seeds and/or other cellular organisms or parts thereof can be grown jointly or separately, and their optimal growth can be ensured by the vertical supply of and periodical exposure to liquid culture medium of pre-planned composition.

The two most important benefits of the connecting tube 3b providing communicating connection between the headspace atmospheres are that as a result of pressure compensation it assists gravitational level control, and furthermore by connecting the atmospheres, it provides the same headspace atmosphere quality in the bioreactor 1 as in the liquid nutrient container 2. Prior art solutions have not envisaged such a headspace atmosphere connection, and catered for pressure compensation (suction) in the bioreactors from the environmental atmosphere, by direct (or perhaps filtered) ventilation. All this heavily reduced the degree of asepticism achievable in the bioreactor.

As shown in the figures, specially designed surfaces, preferably trays hold the inoculated explants and micropropagated offsprings in the bioreactor 1. The separation of the micropropagated offsprings is ensured only by a special, conventionally depicted horizontal vibration and/or orbital shaking mechanism 6. The mechanism 6 can be of a continuously or intermittently operating type.

The micropropagation apparatus is preferably fitted with the controlled lighting 4 directed from the side at the bioreactor 1. Preferably, the connecting tubes 3a, 3b feature the tube joints 7 which can be connected/disconnected in a sterile way also in situ. Therefore, the invention enables the replacement of the liquid culture medium in situ, and furthermore allows the programmed adjustment of the composition of gas atmosphere by direct and indirect methods.

Consequently, the supply of plants and propagules with liquid nutrient in the bioreactor 1 is changed by controlling the interrelated vertical positions of the liquid culture medium container 2 and the bioreactor 1 , by means of the vertically actuating mechanism 5, on the basis of the principle of communicating vessels. As shown in Figs. 1A and 1B, the extreme relative positions of the bioreactor 1 and the liquid culture medium container 2 are regulated by the actuating mechanism 5 via intermittent and/or gradual fine-tuning, ensuring that the plants and propagula are flooded vertically with programmable liquid culture medium as required, in addition to their washing, flushing and/or cultivation in a liquid nutrient of variable height and/or in a liquid culture medium film of highly increased aeration, without having to refill the bioreactor 1 (taken repeatedly to the sterile space) again and again with fresh liquid culture medium to replenish the quantity consumed by the plants.

The orbital shaking and/or vibration mechanism 6 performing the horizontal movement of the bioreactor 1 along its longitudinal axis ensures the programmable horizontal movement of the plant tissues and propagula growing therein, as required, in addition to their separation from each other as required, with or without a liquid culture medium.

In the preferred embodiment shown in Fig. 2, there are Y branches fitted with the taps 8 arranged on the connecting tubes 3a, 3b to allow the simple in situ replacement of the liquid culture medium container 2. Fig. 3 shows that the bioreactor 1 comprises a transparent or only partly transparent cover 9 which can be opened and closed from the top or the side, as well as one or more removable trays 10 which hold the subcultured micropropagation offsprings and plants, and enable the even spreading and separation of plant parts or tissue clusters and propagula during horizontal movement.

The embodiment shown in Fig. 4 is fitted with inlet and outlet gas stubs 11 which enable regulating the composition of the headspace atmosphere in the bioreactor 1. They can be used, for example, in the programmed injection of gases required for micropropagation.

In a given case, the bioreactor 1 may also comprise a passive filter 12 to ensure a natural air change, and the illumination 4 directed at the bioreactor 1 from the side (as a result of the rising hot current generated thereby) may assist the air change by forced current via the passive filter 12 of the bioreactor 1.

In the method and apparatus according to the present invention, of course more than one bioreactor 1 , liquid nutrient container 2, lighting 4, etc. can be applied with joint, group and individual control.

The advantages of the micropropagation apparatus embodying the invention have also been proven by our experimental results. According to these results, in a properly assembled cultivation chamber, the inoculated explants and all further subcultures treated in a programmed way with the liquid culture medium and gases developed much more favourably than in the case of the conventional in vitro cultivation methods.

Examples

Example 1 : Cultivation of tobacco (Nicotiana tabacum cv. Petit Havanna) shoot tip culture Culture conditions: The cultivation period was 5 weeks, from 23 May to 26 June. The cultures grown in the bioreactor were flooded every four hours for 15 minutes by liquid culture medium, and then flushed by the lateral vibration movement of cultivation trays. The leaf and shoot generating cultures were on a plant growth regulator containing culture medium for 6 weeks from 25 October to 5 December, and then they were reinjected into a hormone-free MS culture medium, on which they were grown for another 3 weeks. This period lasted from 5 December to 27 December, after which the culture was evaluated. During this time the explantates developed at an appropriate rate, and therefore the generated shoots reached the appropriate level of development in 5 weeks.

The assessment of shoot tip culture is shown in Figs. 5 and 6. In the bioreactor, the fresh (i.e. wet) mass of plant No. 1 was the largest, and the lowest mass was exhibited by plant No. 6. In the case of solid culture medium, the lowest mass was found in the case of plants Nos. 4, 6 and 7. The masses of plants Nos. 5 and 10 evolved similarly. The last two columns of the diagram show the average of the fresh masses of the plants. The columns indicate that in the bioreactor the individual mass of the plants was 2.1g higher than in the case of using solid culture medium.

Fig. 6 shows that while the fresh mass was more in the bioreactor, the dry mass was slightly lower there than the average of control plants. This is a consequence of the fact that in the bioreactor the plants are continuously in a liquid culture medium, and therefore they are able to absorb more moisture and also emit much more. While the plants in the bioreactor had a fresh mass 2.1g larger than the control plants, the difference between the dry masses was only 0.06g.

Example 2: Sundew (Drosera rotundifolia) shoot culture

Culture conditions: The shoot cultivation period was 5 weeks. The cultures were grown on hormone-free MS culture medium. The cultures grown in the bioreactor were flooded with liquid culture medium for 25 minutes every four hour, and were flushed by the lateral vibration of cultivation trays. During this time, the explantates developed at an appropriate rate, and therefore the shoots generated reached an appropriate level of development in 5 weeks.

The results of the experiment are shown in Figs. 7 to 9. The tests carried out were the following: fresh mass test, dry mass test, examining the number of shoots, measuring the diameters of shoots, measuring the hairiness of shoots, inspecting the roots and performing a pH test. To determine the dry matter, the plants were dried for 24 hours at 105 ⁰ C.

By comparing the fresh and dry masses, it can be determined that liquid culture propagation resulted in a more favourable growth. The water content of plants propagated in a bioreactor was higher. In the case of liquid culture medium, all inspected indicators of the plants (i.e. fresh mass, dry mass, quantity of cultivated plant tissue, moisture) were higher than the growth and development of agar-agar control culture medium. Fig. 7 shows the fresh mass characteristics, Fig. 8 depicts the characteristics related to dry mass, and Fig. 9 is a summary of the main cultivation details.

The invention is of course not limited to the preferred embodiments detailed above, but further versions and modifications are possible within the scope defined by the claims. For example, changing the interrelated vertical positions of the liquid culture medium container and the bioreactor can be achieved by modifying the position of the bioreactor in height or by moving both containers.