WO2008015651A2

WO2008015651A2 - A method for propagating an aquatic plant, a package containing the aquatic plant and a method of displaying the aquatic plant

Info

Publication number: WO2008015651A2
Application number: PCT/IB2007/053044
Authority: WO
Inventors: Helene S. Eriksen
Original assignee: Pixieplant
Priority date: 2006-08-04
Filing date: 2007-08-02
Publication date: 2008-02-07
Also published as: WO2008015651A3

Abstract

A method for propagating a plant (20), in particular an aquatic plant in which a the conventional laboratory method for in vitro propagation of aquatic plants is modified so that the transfer steps between subsequent cultivation vessels with various nutritious culture mediums is modified so that a bag (1) is used as a first cultivation vessel, which is closed and sealed after transfer of a formative tissue part (14), and the proliferation of multiple shoots, the rooting of the shoots and the storage in proceed in the bag (1). The bag constitutes a package, which can be displayed in a convenient manner. Due to the reduced number of transfer steps between different growth environments, healthier aquatic plant can be propagation in vitro in a fast manner and at lower costs than when using the conventional technique.

Description

A method for propagating an aquatic plant, a package containing the aquatic plant and a method of displaying the aquatic plant

The present invention relates to a method for propagating a plant .

The method is of the kind comprising the steps of providing a sterile formative tissue part from a plant to be reproduced, transferring the formative tissue part from the plant to a first sterile cultivation vessel containing a first sterile nutritious culture medium tailored and measured out for propagation of the formative tissue part from the plant and for inducing proliferation of multiple shoots, transferring each shoot to a second cultivation vessel containing a second nutritious culture medium tailored and measured out for propagation and rooting of the shoot, and transferring the rooted shoot for storage in a third cultivation vessel under humid conditions.

An aquarist strives to avoid contamination and infection of the aquarium. By contamination is meant introduction of a substance making the aquarium impure. The contaminant may be living or non-living and poisonous or affect e.g. a culture or a plant in a negative way.

If some of the aquatic plants or all aquatic plants in an aquarium becomes affected by illnesses, dies out or are eaten by fish or damaged by snails the aquarist must substitute the impoverish aquatic plants with new attractive aquatic plants from an external environment to maintain an acceptable ornamental appearance of the aquarium. The same situation and risk of contamination and infection arises if the aquarist just likes to add new aquatic plants to the already existing selection of aquatic plants in his aquarium or if a new aquarium is set up. Every time something new is introduced into the aquarium from an external environment such an introduction may accidentally contaminate the aquarium and infect both aquatic plants and fish.

To minimize this risk, in Denmark many aquatic plants are today propagated from formative aquatic plant tissue and mass produced in a conventional laboratory propagation method under sterile conditions in nutritious test tubes or cups. The major steps of this conventional method involve to provide formative tissue, such as a terminal or axillary bud, from a plant source in a sterile culture environment free of contamination from bacteria and fungi. The formative tissue is often denominated an "explant".

When proliferation of multiple shoots has been induced, the shoots are transferred to fresh media to induce further growth. The further grown shoots are transferred to root in vitro or in soil under non-sterile conditions in flower pots in greenhouses until an appropriate, ready for-sale height has been reached. Each transfer step involve that the plant tissue must acclimate from one environment to the subsequent environment. The potted aquatic plants are sold to the subdealers who e.g. store the pots in display aquariums with the purpose of showing the aquatic plants to the end user, e.g. the aquarist.

This known method involves multiple in vitro and ex vitro transfer steps including transfers of the aquatic plants via and to non-sterile new growth environments. Each transfer may introduce undesired material, e.g. chemicals such as pesticides, algae, snails, fungi, or diseases infectious to the next growth environment .

In addition, although many precautions are taken to avoid contamination in the conventional laboratory method, the many transfer steps increase the risk that e.g. the aquarists without knowing it brings along undesired and contaminating material together with the new aquatic plants, especially from the display aquarium. Hence, the aquarist often finds living organisms, e.g. snails and algae, in his aquarium without having the slightest knowledge of how they where introduced. This is very annoying because these organisms multiply and are very difficult or even impossible to get rid off.

Hence, the aquarist has an unsatisfied need for minimizing this risk and to obtain highly pure, non-contaminating and non-contaminated aquatic plants.

The SacO2 Microsac® is an air permeable bag, commercially obtainable from SacO2, Rozenstraat IA, 9810 Eke, Belgium. The SacO2 Microsac® is recommended for production of mushroom spawn. A depth filtration system in the bag wall forms a barrier against micro-organisms and allows the content in the SacO2 Microsac® to exchange gas with the external environment. A mother culture of pure mycelium is grown on a nutrient broth, glucose-enriched agar medium in a test tube. The grown culture is subsequently transferred to a SacO2 Microsac® containing a substrate for further germination and production of large amounts of mycelium. At the mushroom farms the mycelium are propagated further, preferably in the dark, to produce eatable mushrooms, which is packaged for sale to the consumer via groceries. Also, this production method involves multiple transfer steps. In particular the steps at the mushroom farm and the packing room requires much handling and risk of contamination.

From Japanese patent publication no. JP2002262672 is known a biodegradable, air permeable bag for producing a large amount of plant seedlings, in particular from scions. This bag, which can be expanded to the shape of an ordinary flowerpot, is made of a thin film having a great number of bored, fine pores. Although, such bores allow material to enter the bag, such entrance must be substantially tolerated to allow water condensation to escape, and prevent chlorosis and vitrification and maintain transport of essential substances. Hence, the bag is a non-sterile mini greenhouse. Moreover, green seedlings require a considerable space to grow up and unfold. They are also sensitive to squeezing and crushing. Hence, to produce ordinary plants a spacious, ventilated bag and plenty of storage space is required.

The prior art is tacit about growing aquatic plants in such bags and in fact the major prejudices within the art of propagating aquatic plants has prevented the skilled person from doing so. Such prejudices include that the aquatic plant cannot reach a maturity stage in which the aquatic plant can tolerate to be taken to a non-sterile environment without suffering, and even that the insufficiently matured aquatic plant will die after a very short while. A further prejudice consists in that the aquatic plant will rapidly decompose when confined in a closed bag.

In a main aspect according to the present invention is provided a novel method for producing an aquatic plant, which method comprises less transfer steps at different aquatic plant maturity stages during the propagation and growth process than conventional methods for propagating an aquatic plants .

In a second aspect according to the present invention is provided a method for propagating an aquatic plant, in which method the risk that the produced aquatic plants infects or contaminates the aquarist' aquarium is substantially reduced.

In a third aspect according to the present invention is provided a package for growing and visually inspecting an aquatic plant during growth. In a fourth aspect according to the present invention is provided a package in which an aquatic plant can be solitarily grown through the whole propagation and growth process to be sold directly to the buyer as an off-the-shelf item instead of being transferred several times and at last temporarily stored in a display aquarium as in the prior art.

In a fifth aspect according to the present invention is provided a package containing an aquatic plant, said package takes up less space than a conventional package.

The novel and unique whereby this is achieved consist in the fact that the method comprises, that the formative tissue part from the plant is from an aquatic plant source, the first cultivation vessel is a bag, which is closed and sealed in step b after transfer of the formative tissue part, and the proliferation of multiple shoots, the rooting of the shoots and the storage proceed in the bag, so that all maturity stages and final storage takes place in one and the same cultivation vessel in form of a bag.

The inventor of the present method has demonstrated that an aquatic plant produced with the method according to the present invention is extremely healthy and strong enough to be directly transferred to the aquarium of the aquarist. Because the aquatic plant is propagated and grown in a closed and sealed sterile in vitro environment the aquatic plant is never subjected to infection and contamination and can use all the energy provided by the chemical substances in the nutritious culture medium to increased growth and strengthen the immune system instead of using energy to fight diseases to survive.

The surfaces of any plants (principally leaves and stems) exposed to the air are covered in a wax cuticle that protects the surfaces and prevents the plants from drying out. In the conventional laboratory propagation methods, the aquatic plant tissue are transplanted from an in vitro environment to an ex vitro environment. The aquatic plant has no need for the wax cuticle in a moist environment, but every time the surfaces of the plant are subjected to transfer and are brought into contact with the surrounding atmospheric air the aquatic plant need to build up a wax layer and regenerate new leaves with a wax layer to substitute the leaves which did not survive the transfer. These actions require considerable energy expenditure. When the aquatic plants energy resources are used for generating the protective wax cuticle instead of being used for growing bigger, the many transfer steps causes growth to stagnate from time to time. In the present invention the complete propagation inside the closed, sealed bag keeps the surfaces of the aquatic plant protected at all maturity stages to allow continuous growth without periods of stagnation. Because the plant during all maturity stages is kept suitable wet or moisturized the aquatic plant does not need to build up a protective wax cuticle over and over again at the cost of growth energy. Furthermore, because the aquatic plant is grown up inside a bag with a humid environment, the aquatic plant does not need to readjust to the aquatic environment in the aquarium when planted out.

Accordingly, the aquatic plant produced in a bag using the method according to the present invention is able grow bigger, stronger and healthier in a shorter period of time than aquatic plants produced using the conventional method described above.

The "propagation and growth conditions" may be the same or differ from one aquatic plant to another aquatic plant. In particular the temperature, content and composition of nutrients, pH, air supply, humidity and light supply are adjusted in dependency of the selected aquatic plant and desired time of full maturity. The preferred "formative plant tissue" from an aquatic plant is tissue selected from the group comprising a meristimatic region, a shoot tip, a node or an explant. Meristimatic tissue is made up of small cells capable of dividing indefinitely and giving rise to similar cells or to cells that differentiate to produce the definitive tissues and organs, however the formative plant tissue can within the scope of the present invention just as well be juvenile tissue or ay other kind of immature aquatic plant structures which is able to divide and grow.

When the whole propagation and growth process from formative tissue to final developed mature aquatic plant takes place in one single bag, the aquatic plant can be grown very cost- effective without stressing the aquatic plant during growth.

Moreover, a bag containing an aquatic plant is also easy to transport from place to place, and many bags can be stored at limited storage space.

An aquatic plant dealer does not need to store the many aquatic plants to be sold in an interim aquarium in which the aquatic plants state of health and development are difficult to overview and inspect for the dealer and his customers. When such a conventionally grown aquatic plant is sold, the dealer takes the aquatic plant from the interim aquarium into a conventional, soft plastic bag or rigid plastic cup. In case of a soft plastic bag, the dealer often inflates this bag simply by exhaling into the bag to create a non-compressible structure for storage of the aquatic plant during transport to the end users aquarium. All these transfer steps and changes of environment may contaminate and influence the survival capacity of the aquatic plant. Each time a transfer takes place the aquatic plant must habituate to a new environment, which sets back growth. By using the method according to the present invention, aquatic plants can be produced which, due to their in vitro sterile growth in one single nutritious environment, are very healthy and viable and does not contaminate the end users aquarium. Preferably, the aquatic plant or aquatic plantlet matures inside the bag to a full-grown maturity stage in which the aquatic plant is ready for selling and planting out.

When the nutritious culture medium contains excess sugar in an amount of 2 - 4 %w/v based on the total volume of the nutritious culture medium the aquatic plants becomes independent of its own photosynthesis, with the result that growth can take place with a minimum of light. The preferred sugar is sucrose. The bag serves as a nutritious incubator, which can be stacked in layers on trolleys or on shelves from floor to ceiling in a storage room with only a few light fittings provided in-between.

When at least a part of the bag is made of a transparent plastic film light can penetrate to promote photosynthesis.

Also, the propagation and growth process is very easy to monitor through the transparent plastic bag, which preferably also are clear. If, in spite of all due care is taken, an aquatic plant is infected by microorganisms or becomes unhealthy in another manner, such a bag never find its way to the end user. Such a bag will be disposed immediately.

Suitable plastic films for producing the bag include but are not limited to polypropylen films. As an alternative film material e.g. polyethylene or poly(vinyl chloride) can be used.

In a preferred embodiment for a bag, which is suitable for the present invention, the bag is provided with a filter means, which filter means is permeable for gases and constitutes a barrier to micro-organisms, so that micro-organisms can easily be kept outside the bag. Through the filter means carbon dioxide is allowed to be exchanged with oxygen from the surroundings. The preferred filter means is a depth filter, which are able to retain particles within a filter matrix. The capacity of the depth filter is typically defined by the depth of the matrix and thus the holding capacity for solids. The advantages by using a depth filter is that dehydration of the aquatic plant is effectively avoided, however for some few aquatic plants a surface filter not having a filter matrix is acceptable although not optimum. A suitable depth filter is the depth filter incorporated in the SacO2 Microsac®.

Other commercially available filters can also be used, such as the microfiltration discs produced by TQPL, Brookside, Southern Lane, New Milton, Hampshire, BH257JE, United Kingdom,

In a preferred embodiment for a bag suitable for use in the method according to the present invention the bag is sealingly closed by welding or by means of a slide zipper to prevent contaminants to enter during handling of the bag. If a slide zipper is chosen the end user can easily open the bag without the use of additional tools.

It may also be preferred to provide the bag with identification of the aquatic plant and additional information of how to handle the aquatic plant. Such information may include the final use-by date, and growth instructions including information of how to transfer the grown up aquatic plant to the end users aquarium. A bar code and the price are other useful information.

Advantageously the aquatic plantlet or aquatic plant can according to the present invention be propagated without the use of pesticides. Within the scope of the present invention the term "pesticide" is to be understood as all chemical agents used to control living organisms that are considered to be a nuisance to the propagation of an aquatic plant. It is an advantage to avoid extensive use of pesticides because pesticides are very expensive and can cause harm to the ecosystem.

The invention also relates to a package containing an aquatic plant, said package is obtained by the method according to the present invention.

The inventor of the present invention has discovered that in contrast to ordinary plants and prejudices within the art of propagating aquatic plants, that the aquatic plants can be grown and stored in a confined space without being damaged or suffering. This makes the packages suitable for selling at any retail stage. Because the matured aquatic plant is contained inside its own little green house the aquatic plant is also directly ready for sale via the Internet.

Further, the invention relates to a method of displaying a number of aquatic plants in packages according to the present invention. The method may include that the packages are hung on a hanger or put on a shelve in any shop or supermarket, in order to display the aquatic plant to the consumer in a handy and inspectable manner, without the consumer gets wet as in the conventional way of displaying aquatic plants as described above, and without being afraid of damaging and contaminating the aquatic plants. Preferably the bag is provided with a flap on which the additional information is provided.

Further, the invention relates to a modified method in which only the storage proceeds in the bag. In this modified method the formative aquatic plant tissue is pre-propagated and multiplied in one or more sterile cultivation vessels before being transferred to the bag as in the conventional laboratory method. This method is preferred if the available amount of formative aquatic tissue is very small or if the plant has never been propagated in vitro under sterile conditions before. The method is less expensive than the previously described method because more shoot culture can be propagated before the small shoots are taken to each their bag, with or without filter means, for further propagation.

Further, the aquatic plants produced by the methods according to the present invention can also be used for decorative purposes. E.g. the aquatic plant can be taken out of the bag and planted in a glass bowl, a vase or similar clear container at hand. In this case, the aquatic plant may be sold together with a suitable substrate for promoting growth of the aquatic plant. The substrate may be added to the container. The aquatic plant is taken out of the bag and anchored in the container using decorative pearls or stones. Finally the container is filled with water to provide a decoration.

Slow release nutrients are preferred for some embodiments. E.g. the Osmocote® slow release nutrient products obtainable from Scotts Australia Pty. Ltd., 11 Columbia Way, Baulkham Hills, NSW, 2153, Australia can be incorporated in the bag.

The invention will be explained in greater details below with reference to the accompanying examples and drawing, in which

fig. 1 shows the first propagation method step according to the present invention in which the formative aquatic plant tissue just have been put in the bag,

fig. 2 shows an intermediate growth stage according to the present invention, in which the aquatic plant has begun to mature,

fig. 3 shows an final growth stage according to the present invention, in which the aquatic plant has grown to a full- grown aquatic plant, and fig. 4 shows a number of packages hung on a hanger.

The aquatic plant and nutritious culture medium shown in the drawing are given by way of example. In particular, the indicated specific appearance of the aquatic plant and the texture of the nutritious culture medium are not intended to limit the invention but to enhance understanding of the drawing .

Fig. 1 shows a bag 1 for use in the method according to the present invention.

The bag 1 is made by folding a rectangular blank 2 of a thin, transparent polypropylene film short end to short end. The folded half parts of the blank 2 are the front 3 and the back 4, respectively, of the bag 1. An expandable bottom 5 may or may not be created by simply pleating the area of the blank 2 along a fold line 6. The front 3 and the back 4 are welded together along the opposing lateral edges 7,8 by means of edge weldings 9,10, respectively.

Alternatively, the bag could be made by welding together two separate thin plastic sheets, e.g. polypropylene sheets, together along the lateral edges of the sheets and the bottom edges. In this alternative design the bag has no expandable bottom.

Both ways of making a bag is known to the person skilled in the art and will not be described in further details.

Both the front 3 and the back 4 has a filter means 11, which constitutes a barrier to micro-organisms and is permeable to allow carbon dioxide to escape from inside the bag 1 and oxygen to enter the bag 1. The filter means is made of a plurality of small, absorbing plugs 12, e.g. of cellulose. The filter means 11 is integrated in the bag 1 by any suitable method, e.g. welding.

A substrate 13 in form of a nutritious culture medium 13 is arranged inside the bag 1 to nourish an aquatic plant during growth. The amount of nutritious culture medium 13 indicated in fig. 1 is given by way of example and more or less can be provided in dependency of the requirements of the specific aquatic plant. The nutritious culture medium 13 can include a substance, which serves as an anchor for the roots. Such a substance may just be a gel, such as agar, or mineral wool, such as rock wool. Agar is e.g. polysaccharides extracted from the redalgae Rhodophyceae . The dominating polysaccharides are agarose and agaropectine . Agar is commonly used as solid media for cell culture.

Other materials, having similar anchoring properties, such as lava stones, tree roots, cotton wool or even paper can also be used as the anchor substance.

Basically the preferred substrates 13 are based on Murashige and Skoog 1962 medium, which is a plant growth medium used in the laboratories for cultivation of plant cell culture. (Murashige, T. & Skoog, F. (1962) Physiol. Plant. 15, 473-97) The substrate is further enriched with nutrients, including one or more energy sources, vitamins, minerals and hormones, and any other agents that may have been found to be vital or advantageously to the continued growth and well-being of the aquatic plant. An excess amount of sugars of 2 - 4 %w/v based on the total volume of the nutritious culture medium is included as the energy source in the nutritious culture medium, so that the growing aquatic plant has sufficient energy for metabolism, maintenance of vital cellular activity, respiration, and air circulation and continued growth. Also, the pH of the nutritious culture medium is adjusted to provide the optimum growth condition of the aquatic plant to be propagated and grown in the bag.

The bag, including the nutritious culture medium, is sterilized and stored under sterile condition until use. Alternatively a sterile the nutritious culture medium is added to the bag.

As is seen in fig. 1, sterile formative tissue 14 from an aquatic plant source is introduced in the bag 1 through an opening 15 opposite the bottom 5 of the bag 1 under sterile conditions, e.g. in a Laminar Air Flow (LAF) bench in which a stream of filtered-sterilized, bacteria- and spore-free air constantly blows. The sterile formative tissue 14 is selected to have a node 15 for further propagation, and is placed into or in contact with the sterile nutritious culture medium 13, so that the various parts of the aquatic plant can start to grow up.

The formative tissue may be pre-propagated and multiplied in one or more sterile cultivation vessels before being transferred to the bag 1 or be directly introduced into the bag 1. After introduction, the bag 1 is subsequently closed by welding the top edges 16,17 of the bag 1 together.

The bag 1, including the formative tissue 14, is now left under growth conditions suitable for propagating and growing an aquatic plant from the specific aquatic plant source. In particular the temperature and the light are adjusted to promote further growth.

The bag 1 serves as a very effective incubator. After only a short while, typically 1- 2 weeks, a small aquatic plantlet 18 has grown up, as indicated in fig. 2. The aquatic plantlet 18 can exchange gas with the external environment through the filter means 11, and is kept isolated from contaminants. The filter means 11 also provide for appropriate humidity inside the bag 1 so that drying out of both the aquatic plantlet 18 and the nutritious culture medium 13 is effectively prevented and initial rooting 19 is seen. Due to the filter means a relative humidity of between 60 - 80 % can be maintained inside the bag, in order to facilitate and enhance nutrient uptake and prevent necrosis and hyperhydricity .

The aquatic plantlet 18 stays in the bag 1 under appropriate growth conditions and is allowed to grow larger, e.g. to the maturity stage and the height shown in fig. 3 in which a well- developed full grown aquatic plant 20 with a fully developed rooting 21 is indicated inside the bag 1. The bag 1 with the aquatic plant 20 is now ready for selling.

The mature aquatic plant 20 is very healthy and un-stressed because both the starting tissue, i.e. the formative tissue, and the aquatic plantlet during all its propagation and growth stages are left in peace.

Suitable growth conditions and compositions and kinds of the nutritious culture medium may be determined by way of experimentation and subsequently tabulated. As a starting point for producing such a table, facts from the nature or from the conventional propagation methods can be used and adapted to the method of the present invention.

As seen in fig. 4 the bag 1 can be provided with suspension means 22 and a flap 23 with e.g. any information 24 that the aquarist may need for growing the aquatic plant 20 in his own aquarium. Both the bag 1 in itself or the bag 1 provided with suspension means 22 and information flap 23 can be seen as a package 25 within the scope of the present invention. In fig. 4 four packages 25 are hung on a hanger rod 26. The hanger rod protrudes from a mounting 26 on a wall (not shown) . The four packages 25 are very easy to overview for the aquarist or any other consumer. Sick aquatic plant is easy to dispose without having been in contact with the other aquatic plants .

Examples

Example 1: Production of Hermianthus micranthemoides

Shoot tips having a length of about 4 mm were cut with a sterile scalpel from a Hermianthus source. The shoot tips were washed in tap water, and then sterilized for 90 seconds using mercury chloride [Ih w/v HgCl₂] as the disinfectant. The sterilized shoot tips were rinsed three times with distillate water and transferred to a culture dish in a LAF bench and tested for sterility.

When sterility was confirmed the shoot tips were transferred to a bag according to the present invention. The bags were equipped with depth filters and were one-quarter filled with a substrate mixture composed of 50 ml Murashige and Skoog, 1962 medium. 3%w/v sucrose based on the total volume of nutritious culture medium and agar was added to the bag, as additional basis for photosynthesis and for fixating the shoot tip, respectively. Further sucrose and agar assists in keeping the osmotic pressure at a level, which prevents dehydration and negative influence on the nutrient uptake. The substrate mixture has pH 5,7.

In a LAF-bench procedure the sterile shoot tip is put in the bag in contact with the substrate under sterile conditions.

The bag is closed by welding. The closed bag is kept under light conditions, corresponding to the light condition used in the conventional methods, at 23°C for six weeks after which period a ready-for-sale aquatic plant has grown up.

In the conventional laboratory method, in which the aquatic plantlet is transferred several times and repotted, the same final maturity stage is reached after 12 weeks. In addition, when using the method according to the present invention only half the conventional amount of mineral components of the

Murashige and Skoog, 1962 medium is needed for in vitro generation of Hermianthus micranthemoides .

Example 2 Production of aquatic plants of the genus Microsorium spec.

Sterile meristems from Microsorium were obtained. The meristems had a length of about 2 mm and is produced and sterilized using 1 %v/v sodium hypochlorite (NaClO) for 5 hours instead of mercury chloride, otherwise using the procedure described in Example 1.

The substrate differs from the substrate defined in Example 1 only in that the concentration of sucrose is 2 %w/v instead of 3%w/v, and that 500 mg of a 2- (N-morpholine) ethane sulfonic acid buffer (MES) per liter nutritious culture medium in added to keep pH at 5,7 during the very slow maturity process.

In a LAF-bench procedure the sterile meristem was put in the bag in contact with the substrate under sterile conditions. The bag was closed by welding. The closed bag was kept under light as describe for Example 1 at 26°C for twelve weeks after which period a ready-for-sale aquatic plant has grown up. The same maturity stage is reached after 6 month when using the conventional laboratory method.

The size of the bag is chosen in dependency of the size of the mature full-grown aquatic plant. Suitable size for small aquatic plants are 9 cm x 16 cm and for larger aquatic plants 13 cm x 25 cm, but any size is within the scope of the present invention .

The method according to the present invention is much faster than the conventional laboratory method described above in which the aquatic plantlet must be transferred between a number of cultivations vessel several times and repotted in a nursery. Also, the inventive method only requires a quarter of the storage space used in the conventional method. The risk of contamination and infection is almost completely eliminated. Chemicals other than required to support growth are not used. In the conventional aquatic propagation method in the nursery the potted aquatic plants are irrigated all the time resulting in a huge waste of water and vast energy consumption. Moreover conventionally the aquatic plants are treated with pesticides and retardants through the propagation.

The aquatic plant propagated using the inventive method is always kept in a wet environment, which is natural for aquatic plants and the plant at any maturity stage is more robust and vital than the aquatic plant propagated using the conventional laboratory method.

In summary, production of aquatic plant using the in vitro propagation method according to the present invention are fast and can be done at lower costs than when using the conventional technique.

The produced aquatic plants are very clean. No vermins, insects, bacteria, fungi, or snails are observed, and use of pesticides and retardants are eliminated. In addition a high rate of growth is observed because juvenile aquatic plants has a faster growth than adult plants.

Claims

1. A method for propagating a plant (20), comprising the steps of a. providing a sterile formative tissue part (14) from a plant (20) to be reproduced, b. transferring the formative tissue part (14) from the plant (29) to a first sterile cultivation vessel (1) containing a first sterile nutritious culture medium (13) tailored and measured out for propagation of the formative tissue part (14) from the plant (14) and for inducing proliferation of multiple shoots, c. transferring each shoot to a second cultivation vessel containing a second nutritious culture medium tailored and measured out for propagation and rooting of the shoot, and d. transferring the rooted shoot for storage in a third cultivation vessel under humid conditions, characterised in that the method comprises, that - the formative tissue part (14) from the plant (20) is from an aquatic plant source (20), the first cultivation vessel (1) is a bag (1), which is closed and sealed in step b after transfer of the formative tissue part (14), and - the proliferation of multiple shoots in step b, the rooting of the shoots in step c and the storage in step d proceed in the bag (1) .

2. A method according to claim 1, characterised in that the formative tissue part (14) from the aquatic plant source

(20) is selected from the group comprising a meristimatic region, a shoot tip, a node or an explant.

3. A method according to claims 1 or 2, characterised in that at least a part of the bag (1) is made of transparent plastic film.

4. A method according to any of the preceding claims 1, 2 or 3, characterised in that the bag (1) is provided with a filter means (11), preferably a depth filter means or a surface filter means, which filter means (11) is permeable for gases and constitutes a barrier to microorganisms .

5. A method according to any of the preceding claims 1 - 4, characterised in that the bag (1) is sealingly closed by welding or by means of a slide zipper.

6. A method according to any of the preceding claims 1 - 5, characterised in that the bag (1) is provided with identification of the aquatic plant and additional information of how to handle the aquatic plant.

7. A method according to any of the preceding claims 1 - 6, characterised in that the aquatic plantlet (18) or aquatic plant is propagated without the use of pesticides .

8. A package (1;25) containing an aquatic plant, said package is obtained by the method according to any of the preceding claims 1 - 7.

9. A method of displaying a number of aquatic plants (20) or aquatic plantlets (18) in which a number of packages 1;25) according to claim 8 are hung on a hanger (26,27) or stacked on a shelf.

10. A method according to any of the preceding claims 1 - 7, modified in that only the storage in step d proceed in the bag (1) .