ZA200302000B

ZA200302000B - Culture system.

Info

Publication number: ZA200302000B
Application number: ZA200302000A
Authority: ZA
Inventors: Louis Johan Wagenaar
Original assignee: Louis Johan Wagenaar
Priority date: 2000-09-22
Filing date: 2001-09-24
Publication date: 2004-03-12

Description

CULTURE SYSTEM

The present invention xelates to a culture system for plants and/or plant tissues.

It is known to make use of closed culture systems, for instance for reproducing, shoot forming, sowing and cultivating young plant material, root crop material ox plant tissues, wherein the nutrients reguired for the plant material, such as salts and carbohydrates, are generally held in sterile systems in semi-solid feed medium such as agar.

For the culture of plants or plant tissues the providing and sustaining of the correct growth conditions are of very great lmportance. Not only do temperature, light intensity and the presence of molsture play an important part, the presence and composition of the correct nutrients are also extremely important for optimum development of the plant material.

Because diffusion of nutrients in semi-solid feed medium or soil is difficult, when such culture media are used the feed of nutrients not available in the immediate vicinity of the roots, cut surface or other take-up point of the plant becomes more difficult as the distance which must be bridged by the nutrients to for instance the root becomes larger. The waste matter generated by the plant material simultaneously accumulates around the plant material, whereby the optimum development of the plant material is inhibited. Furthermore, the nutrient requirement is seldom constant during the growth process, and often increases through time. The continuous feed of sufficient nutrients is therefore of great importance for the development of the plants and/or plant material.

The use of a liquid culture medium instead of for instance agar partly obviates these problems. One of the problems of using a liquid culture medium is however that there is a maximum osmotic value for the culture medium because the plants or tissueg for cultivating accept only

CWO 02/37950 PCT/NLO1/00700 a determined maximum osmotic pressure (often differing per variety).

During the development of the plant material nutrients are taken up from the culture medium and waste products are relinguished to the culture medium. The guantity of nutrients present in the culture medium therefore decreases through time. The addition of new nutrients during culture is not generally desirable ang, in the cape of closed gystems, is moreover difficult and time-conguming. It is further generally desirable to keep the culture system sterile, and additional wanipulations of the culture system, such as adding extra nutrients, increase the chance of infections in the culture.

The object of the present invention is to provide a culture system wherein the above gtated problems are reaolved.

This objective ig achieved with the invention by providing a culture system for plants and/or plant tissues, comprising at least one culture medium comprising one or more nutrients and a stock of one or more nutrients from which the nutrients are dispensed gradually to the culture medium. Thus is ensured that a sufficient quantity of nutrients is available in the culture system during the whole of the culture without additional manipulations of the culture system, such as adding new nutrients after a period of time, being necessary. In the case of for instance sterile culture systems, there is hereby legs chance of compromising the sterility.

A gradual dispensing according to rhe present invention is understood to mean that the nutrients axe relinquished to the culture medium not immediately after placing of the stock, but gradually. In this manner the quantity of nutrients remains continuously optimal, and it is possible to prevent a temporary, possibly harmful increase in nutrients occurring in the culture medium.

In a particular embodiment the culture system comprises a stock of one or more nutrients from which the

. nutrients are dispensed in delayed manner into the : culture medium. This means that at the start of the : culture the nutrients are dispensed not immediately but : only after a determined time, i.e. with a ndelay®", to the “ culture medium and become available to the plants/tissues in the culture system. Directly at the start a high concentration of nutrients can hereby be added to the © culture system without the tender young plants and/ox plant tissues coming into contact with this high concentration of nutrients and being harmed thereby. When after a time the nutrients are then dispensed in oo - "delayed? manner to the culture medium, the concentration of disgolved nutrients in the culture medium has already

Co decreased, thus preventing too high a concentration.

Furthermore, the plants are generally less vulnerable at that stage of the culture.

The nutrients are preferably dispensed in delayed and gradual manner into the culture medium.

Tn order to ensure that for instance waste products do not accumulate around the plant material, in a no suitable preferred embodiment of the culture system according to the invention the culture medium is a liquid culture medium, The culture medium can herein be any appropriate culture medium. : "In another suitable embodiment the culture medium is a liquifying medium. The medium for instance } comprises herein substances with the particular property that they change in structure and/or Yiscosity when there Co is a change in determined ambient factors, such as for instance the pH and/or the salt concentration of the medium. Substances suitable for this purpose are, among ' others, Gelxite, carbomer, poloxamer and/or coconut Oil ' derivatives with linked polar head. ‘such a liquifying . : medium enables dispensing of larger guantities pf nutrients to the culture, The nutrients can then be released gradually by .changing for instance the pH or salt concentration; and thereby the consistency of the . medium. During the liquifying the nutrients

AMENDED SHEET 2004 -g4- 2g

N WO €2/37950 . PCT/NL01/006700 : gradually become available to the culture. Up until that moment they will have been present in for instance the geni-solid medium, wherein the osmolarity has no adverse effect on the plant growth. Another advantage of a liquifying medium is that, when such a medium is used instead of a liquid medium, it is possible to prevent tissues and/or small plants "drowning? in the culture medium. In the initially {semi-) solid medium the plants/tissues still remain (for the greater part) above . . 10 the water level. As the culture medium liquefies in the course of time, the plants have meanwhile grown, : so that there is less chance of drowning.

The term "nutrient stock” as used in the present invention relates to a "carrier" in/on which a concentrate of nutrients is present. The nutrients are herein present in a concentration higher than the concentration of the nutrients in the culture medium irself. The term "nutrients" as used in the present invention relates to substances which are added to the culture medium and can be taken up therefrom by the plant.

The gtock can be embodied in any suitable manner such that the nutrients can be placed in highly concentrated form in for instance a liquid culture medium, whereafter the nutrients are relinquished gradually, and preferably in delayed manner, from that - - stock to the culture medium and can be taken up by the - plant material. The nutrient stock preferably comprises a . : tablet, block, capsule, granulate, moulded product, powder mixture, paste or syrup. In a particular preferred embodiment the nutrient stock, such as for instance the tablet, the moulded product, block or granule, is coated for even better regulation of the delayed, gradual dispensing of the nutrients. During the coating the same substances can be used as those of which the stock consists, although other substances can also be used for this purpose.

AMENDED SHEET vue -04- 29

A particularly suitable embodiment of the culture system according to the invention is obtained when the stock comprises at least one or more fatty acids and/ox derivatives thereof, in which the nutrients are 5 incorporated in finely distributed manner. Owing to The specific properties of Fatty acids a stock is obtained which is not water-soluble but which is however water- permeable up to a certain point. The aqueous culture medium is largely retained by the aliphatic chains of the \ 10 fatty acids, while a swall amount of the culture medium can still permeate into the moulded product due to the polar head of fatty acids. The nutrients present in for instance the moulded product consisting of fatty acids are thus taken up in delayed gradual manner into the acqueoug culture medium.

According to the invention the fatty acide and/or derivatives thereof preferably have a melting temperature which is above the temperature of the culture wedium (generally arcund 15-20°C), so that the stock does not dissolve too quickly into the culture medium. The fatty acids and/or derivatives thereof preferably have a melting temperature lying above 15°C, in particular above 20°C,

Diverse fatty acids and/or fatty acid mixtures can be used for the culture system according to the invention, such as for instance stearic acid, palmitic acid and/or myristic acid. A very suitable fatty acid fox use in the invention is stearic acid. It is algo possible to prepare mixtures of fatty acids, some fatty acids of which exist as oil below the temperature of the culture medium, such as linolenic acid or oleic acid. Oils, fatty alcohols, lecithins or waxes which are liquid or semi- solid at the temperature of the culture medium can also be included or serve as basis. In order to cbtain a gradual dispensing, fatty acid derivatives can also be used, wherein for instance one or more molecules are linked to the fatty acid, such as ethyl oleate, sodium cetyl stearyl sulphate, polyethylene glycol ethers of

& fatty acids or fatty alcohols, or magnesium stearate. For instance the melting point and/or the structure and/or the water permeability of the product can hereby be changed or regulated.

In addition to fatty acids and/or derivatives thereof, the nutrient stock can further comprise one or more other suitable compounds from which the nutrients are dispensed gradually, such as for instance polymers i such ag (hydroxyethyl) cellulose and other celluloge : 10 derivatives, polysorbates, sorbitan fatty-acid esters, wool wax and other (synthetic) waxes, cellulose acetates, polyacrylates, polyvinyl acetates, shellac, carbomers, poloxamers and/or polymerized coconut oil fatty-acid derivatives, and blodegradable polymers such as pelylactice acids, polygalactides and wixtures hereof.

In a very suitable embodiment of the invention the stock comprises a moulded product of stearic acid in which the nutrients are incorporated in finely distributed manner.

Tn another advantageous embodiment of the invention the stock comprises one or more compounds which after dispensing into the culture medium are converted into one or more nutrients. This ensures that the nutrients are relinquished in gradual manner to the culture wedium. The conversion of the couwpounds into nutrients can take place for instance under the influence of enzymes.

The nutrients which can be used for the nutrient stock in the culture system according to the invention preferably comprise carbohydrates, hormones, proteins, vitamins, plant growth regulators, crop protecting agent, salts and/or minerals and/or combinations thereof, In addition, it can be advantageous to add one or more pesticides/herbicides, optionally in combination with the nutrients.

The carbohydrates preferably comprise starch, saccharose, maltose, glucose, fructose, oligosaccharides and/or (malto)dextyxins. The advantage of using carbohydrates with longer chains is that the osmolar pressure of the culture medium is not thereby increased.

The hormones which can be used in the culture system according to the invention comprise for instance auxins, gibberelling, abscisinic acid and/or cytckinins, such as benzyl-amino-purine (BAP) and kinetin. \ Suitable salts which can be used in the culture \ system according to the invention are for instance \ phosphate, nitrate, magnesium, potassium, berate and/ox sulphate salts.

In a preferred embodiment of the culture system the nutrient stock is arranged directly in the culture medium. The stock can however also be used in any other appropriate manner in the culture system. It can for instance be suspended above the culture medium, wherein the nutrients drip gradually out of the stock intc the culture system, or be fixed to the wall or cover from which the stock is released after a time, i.e. in delayed manner, and drops into the culture medium so that the nutrients become available.

The invention further relates to and provides a nutrient stock as described above for use in a culture system for plants and/or plant tissues. In particularly suitable manner the stock according to the invention can be applied in a culture system as described above. The nutrient stock can however also be used for plants in other known culture systems for plants, such as for instance in cultivation of plants in open ground, in hydrocultures, root cultures, so-called hydropollex or for instance systems with rockwool.

The nutrient stock can further be used in suitable manner in a plant or bloreactor or simpler variants thereof. Plant parts such as cell cultures, cell suspensions and root cultures are herein fox instance placed and cultivated in a reactor, wherein the nutrients are dispensed for instance gradually to the culture medium by means of a nutrient stock according to the invention,

The invention is further elucidated with reference to the accompanying examples and figures.

Figures 1-4 show dispensing .patterns of the nutrients from diverse nutrient stocks according to the invention. The amount of released nutrients (% of maximum) is herein plotted over time.

Figure 1 shows the dispensing pattern of a coated moulded product of stearic acid incorporating saccharose (60% stearic acid/40% saccharose).

Figure 2 shows the gradual dispensing pattern of an uncoated moulded product of stearic acid incorporating both BAP (50 pg) (indicated with asterisks) and sugar (50%) (indicated with dots).

Figure 3 finally shows the dispensing pattern of a moulded product of a mixture of stearic acid and palmitic acid incorporating 30% saccharose.

Figure 4 shows the dispensing pattern of a tablet made from equal parts of saccharose and maltose incorporating 0.1 mg kinetin and 10% PEG, whexein the tablet is coated with a solution of high-molecular polylactic acid.

EXAMPLE 1

Potato tissue was cultured in a culture medium with homogeneously dissolved nutrients (3% saccharose, 100 pug/1l BAP and DEW) and a delayed dispensing salt in the presence and absence of a sugar stock.

Moulded: products of stearic acid comprising 40% saccharose were coated with a coating of stearic acid.

Four of such coated moulded products of 5 gram (total 20 grams) were added to 150 ml of aqueous medium with a standard salt and sugar composition sufficient for a month's growth of the tissues. The sugar was dispensed gradually from the moulded products with a delay of about a week and taken up into the culture medium. An average of about 10% of the total quantity of sugar was taken up per week in the aqueous culture medium. The dispensing speed was additionally delayed, particularly in the first three weeks, by the coating.

The test set-up is summarized in table 1.

Table 1. Experiment 1 \, \ delayed dispensing salt product

CE CR LE

In the case of the plant tissue cultured in the presence of the nutrient stock from which sugar was dispensed gradually in delayed manner to the culture medium, 30-70% more leaf pairs and 30-70% more formed shoots were observed after 6 weeks.

EXAMPLE 2

In a subsequent experiment lilles were cultured in a culture medium with dissolved sugar, salt and BAP in the presence and absence of a coated nutrient stock according to the invention comprising 30% sugar and hormone (500 pg). The test set-up is summarized in table 2.

Table 2, Experiment 3

CS CR CR

10 20-70% more bulb formation was seen in the culture to which the coated sugar and hormone stock were added.

EXAMPLE 3

The dispensing pattern of diverse nutrient stocks according to the invention was determined. The results hereof are shown in figures 1-4.

Figure 1 shows the dispensing pattern of a coated moulded product of stearic acid incorporating saccharose (60% stearic acid/40% saccharose). As becomes apparent from the figure, the sugar is relinguished gradually to the medium in delayed manner, i.e. with a delay of about 1 week,

Figure 2 shows the gradual dispensing pattern of an uncoated moulded product of stearic acid incorporating both BAP hormone (50 ug) and sugar (50%).

Figure 3 finally shows the dispensing pattern of a moulded product of a mixture of stearic acid and palmitic acid incorporating 30% saccharose in stearic acid, 3Q Figure 4 shows the dispensing pattern of a tablet : made from equal parts of saccharose and maltose incorporating 0.1 mg kinetin and 10% polyethylene glycol, wherein the tablet is coated with a solution of high- molecular polylactic acid,

Claims

' CLAIMS

' 1. Culture system for plants and/or plant tissues, comprising at least one culture medium comprising one ox more nutrients and a stock of one or more nutrients from which the nutrients are dispensed gradually to the culture medium.
2. Culture system as claimed in claim 1, characterized in that the nutrients are dispensed in delayed manner to the culture medium.
3, Culture system as claimed in claim 1 or 2, characterized in that the nutrients are dispensed in delayed and gradual manner to the culture medium.
4. Culture system as claimed in claim 1, 2 or 3, characterized in that the culture medium is a liquid culture medium.
5. Culture gystem ag claimed in any of tthe claims 1- 4, characterized in that the culture medium is a uniformly spreading medium.
6. Culture system as claimed in any of the claims 1- 5, characterized in that the nutrient stock comprises a tablet, block, capsule, granulate, moulded product, powder mixture, paste or syrup.
7. Culture system as claimed in any of the claims 1- 6, characterized in that the stock is coated.
8. Culture system as claimed in any of the claims 1- 7, characterized in that the stock comprises at least one or more fatty acids and/or derivatives thereof, in which the nutrients are incorporated in finely distributed manner,
} 9. Culture system as claimed in claim 8, characterized in that the fatty acids and/ox derivatives thereof have a melting temperature which is above the temperature of the culture medium.
10. Culture system as claimed in claim 9, characterized in that the melting temperature lies above 15°C.
11. Culture system as claimed in claim 2 or 10, ’ characterized in that the melting temperature lies above 20eC. *
12. Culture system as claimed in any of the claims 8-11, characterized in that the fatty acids comprise stearic acid, palmitic acid and/or myristie acid.
13. Culture system as claimed in claim 12, characterized in that the fatty acid is stearic acid.
14. Culture system as claimed in any of the claims 6-13, characterized in that the stock comprises a moulded product of stearic acid in which the nutrients are incorporated in finely distributed wanner.
15. Culture system as claimed in claim 14, characterized in that the stock is coated with stearic acid.
16. Culture system as claimed in any of the claims 1-15, «haracterized in that the stock comprises one or more compounds which after dispensing into the culture medium are converted into one or more nutrients.
17. Culture system as claimed in any of the claims 1-16, characterized in that the nutrients comprise carbohydrates, hormones, proteins, vitamins, plant growth regulators, salts, minerals and/or cxop protecting agent.
18. Culture system as claimed in claim 17, characterized in that the carbohydrates comprise starch, saccharose, maltose, glucose, Eructose, oligosaccharides and/or {(malto)dextrins.
19. Culture system as claimed in claim 17, characterized in that the hormones comprise auxins, gibberellins, abscisinic acid and/or cytokinins.
20. Culture system as claimed in claim 17, characterized in that the salts comprise nitrate, phosphate, magnesium, potassium, borate and/or sulphate salts.
21. Culture system as claimed in any of the claims 1-20, characterized in that the stock is arranged in the culture medium.
22, Nutrient stock for use in a culture system for plants and/or plant tissues.
23. Stock ag claimed in claim 22, characterized in ' that the stock comprises a tablet, block, capsule, granulate, moulded product, powder mixture, paste or Syrup.
24. Stock as claimed in claim 22 or 23, characterized in that the stock is coated.
25. Stock ag claimed in claim 22, 23 or 24, characterized in that the gtock comprises at least one or more fatty acids and/or derivatives thereof, in which the nutrients are incorporated in finely distwxibuted manner,
26. Stock as claimed in claim 25, characterized in that the fatty acids and/or derivatives thereof have a melting temperature which is above the temperature of the culture medium.
27. Stock as claimed in ¢laim 25 or 26, characterized in that the melting temperature lies above 15°C.
28. Stock as claimed in claim 25, 26 or 27, characterized in that the melting temperature lies above 20°C.
29. Stock as claimed in any of the claims 25-28, characterized in that the fatty acids comprise stearic acid, palmitic acid and/or myrisgtic acid.
30. Stock as ¢laimed in claim 29, characterized in that the fatty acid is stearic acid.
31. Stock as claimed in any of the claims 23-30, characterized in that the stock comprises a moulded product of stearic acid in which the nutrients ave incorporated in finely distributed manner,
32. Stock as claimed in claim 31, characterized in that the stock 1s coated with stearic acid.
33. Stock as claimed in any of the claims 22-32, characterized in that the stock comprises one or more compounds which after dispensing into the culture medium are converted into one or more nutrients.
34, Stock as claimed in any of the claims 22-33, characterized in that the nutrients comprise carbohydrates, hormones, proteins, vitamins, plant growth , regulators, crop protecting agent, salts and/or minerals.
35. Stock as claimed in claim 34, characterized in that the carbohydrates comprise starch, saccharose, maltose, glucose, fructose, oligosaccharides and/or (malto)dextrins.
316. Stock as elaimed in claim 34, characterized in that the hormones comprise auxing, gibberellins, abscisinic¢ acid and/or cytckinins.
37. Stock as claimed in claim 34, characterized in that the salts comprise nitrate, phosphate, magnesium, potassium, borate and/or sulphate salts.