GB2204591A - Method of preparing an algae culture and a culture medium - Google Patents

Abstract

Suspension culture of seaweed especially the genus Laminaria is prepared by surface sterilizing seaweed stipe of at least 10cm long, washing, removing the stipe surface; and then inoculating agar based culture medium with segments of the sterilized stipe. After incubation to induce a callus growth, cells therefrom (preferably gametocytes) are introduced into a liquid culture media and incubated thereby providing a suspension culture. The culture medium comprise standard inorganic salts, optional vitamins, and is characterized by the inclusion of phytohormones and the use of filter-sterilized seaweed.

Description

Method of Preparing an Algae Culture, and Culture Medium used Hereto This invention relates to culture of multicellular algae, particularly of the division Phaeophyta and especially, but nowt exclusively, of the genus Laminaria Whereas tissue culture techniques have been developed successfully for higher plant material, such techniques have not been readily applicable to seaweeds. At the present time any attempt to apple these known techniques to culture of such tissues has not proved to be entirely satisfactory from a commercial viewpoint. Even the most successful of these, callus culture growth, has proved undesirably slow. Some workers (Fries, L., 1980. J. Phycol. 16.

475-477; Saga, No., & Sakai, Y., 1977. Bull. Jap. Soc.

Phycol. 25 Supp. 297-301; Saga et al, 1978. Bull. Jap.

Soc. Sci. Fish. 44 (1) 87; Yan, Z., 1984. Hydrobiologia 116/117. 314-316) have succeeded in producing callus cultures of Phaeophyta, but there is no evidence in the literature of suspension culture ever having been successfully applied to Laminaria sp or similar algae.

There is, however, a strong demand for a solution to the problems encountered with culture of such tissues to be found. A number of important substances are produced by the Phaeophyta including lectins and alginates. The latter have a major role both in the food and pharmaceutical industries. Brown seaweeds are also used for human consumption, and recently their use has been extended to include production of methanol and methan fuel from biomass. Products are extracted from seaweed which is either collected from the natural environment or artificially cultivated and propagated.

this is generally by one of three methods of mariculture, the floating raft, the casting stone with sporeling', and the submerged floating rope techniques.

Such methods are exploited mainly in China and Japan, and there are problems in ensuring continuous biomass production throughout the year. (Brinkhuis et al..

1983.J. World Maricult. Soc. 14. 360-379; 1984.

Hysrobiologia 116/117, 259-262).

A method of culturing Phaeophyta without the problems associated with mariculture or collection, of seasonal variation, contamination and variation between batches would be advantageous, especially in exploitation of by-products such as alginates. In these a uniformity of product quality and composition is highly relevant to their further use.

Accordingly it is an object of this invention to obviate or mitigate the aforementioned problems.

The invention provides a method for preparing novel a suspension culture of seaweed, especially phaeophyta and particularly the genus Laminaria,such as Laminaria dipitata and Laminaria sacharina. Novel culture media have been prepared which are especially suitable for preparing the suspension cultures of Laminaria, but are also useful in the culture of other algae.

Thus, according to a first aspect of the present invention there is provided an algae culture medium comprising major inorganic salts, minor inorganic salts and phytohormones made up in filter sterilized sea water.

Major and minor in respect of the inorganic salts are known terms in the art and refer to quantities of the salts.

The algae culture medium is advantageously based (substantially) on a known medium which is in general use for culturing of marine microalgae (Guillard, R.R.L., 1963. In Culture of Marine Invertebrate Animals ed. W.D. Smith, M.H. Chanley pp. 29-61.

Plenum Press, New York) but which has been modified to provide several novel characteristic features, namely the inclusion of phytohormones, and the use of filter-sterilized sea water. Vitamins are preferably included in the culture medium. The phytohormones are preferably selected from 2,4 Dichlorophenoxy-acetic acid, kinetin, and Indole acetic acid, in a concentration of from 2-6mg per litre of filtersterilised sea water. The major inorganic salts as described on the above known medium are at a concentration of 100 to 200mg per litre of filtersterilised sea water and selected from Also3, NaH2PO4.

2H20, NH4cl, and Na2SiO3.9H20; the minor inorganic salts are trace elements and ferric sequestrene of concentrations respectively of from 0.15 to 0.25mg and from 0.25 to 0.75 mg per litre of filter-sterilized sea water, the trace elements being selected from CuSO4 5H20, 2nS04. 7H20, CoCl2.6H20, Mncl2.4H2O and Na2M0O4.

2H20. When vitamins are present preferably they are are a concentration of from 0.05 to 0.15 mgl and selected from biotin, vitamins B12, thiamine Hcl. The above culture media is suitable for a suspension culture.

Additionally, the above media may further comprise agar in an amount of about 6.0gel~1 to provide a plate culture medium. For example, in the following Table a preferred medium is shown for both forms of culture.

TABLE PLATE SUSPENSION COMPONENT CULTURE CULTURE NaNO3 . 75.Omgl-1 75.Omgl-1 NaH2PO4.2H2O 5.6mgl-1 5.6mgl-1 NH4Cl 26.5mgl-1 26.5mgl-1 Na2SiO3.9H2O 30.0mgl-1 30.0mgl-1 Cu SO4.5H2O 0.0098mgl-1 0.0098mgl-1 Zn SO4.7H2O 0.0220mgl-1 0.0220mgl-1 CoClz.6H2O 0.0100mgl-1 0.0100mgl-1 MnCl2.4H2O 0.1800mgl-1 0.1800mgl-1 Na2MoO4.2H2O 0.0060mgl-1 0.0060mgl-1 Ferric sequestrene 0.0050mgl-1 0.0050mgl-1 Biotin 0.0005mgl-1 0.0005mgl-1 Vitamin B12 0.0028mgl1 0.0028mgl-1 Thiamine HCl 0.1000mg-1 0.1000mg-1 2,4 Dichlorophenoxyacetic acid 2.0mgl-1 2.0mgl-1 Kinetin 0.3mgl-1 0.3mgl-1 Indole Acetic acid 3.Omgl-1 3.Omgl-1 Agar 6.Ogl-1 Filter Sterilized Sea Water 1 litre 1 litre pH 8.0 8.0 The culture media as defined in the first aspect is especially suitable for preparing a novel inoculum of seaweed and a novel suspension seaweed culture from the inoculum. As already mentioned the method is especially applicable to the Laminaria genus.

Accordingly in the second aspect of the invention there is provided a method of preparing an inoculum for a seaweed suspension culture comprising obtaining a seaweed stipe which is at least 10cm long; surface sterilizing the stripe in an appropriate sterilant; washing the stipe under sterile conditions; removing the surface layer of the stipe; inoculating under sterile conditions, an agar based culture medium with cells from said sterile stipe; and incubating the inoculated medium under conditions favourable for callus growth.

Preferably the agar based culture medium is inoculated with tissues selected from the meristematic or region adjacent thereto of the scerilized stipe.

From this inoculum, a seaweed suspension culture is prepared, which comprises inoculating a culture medium (agar free -as defined in the first aspect buthaving the phytohormones as an optional and preferred component) wtith seaweed cells obtained from a callus growth induced according to the second aspect of the invention, and then incubating the suspension culture under favourable growth condition.

The suspension culture is desirably aerated.

Perferably also, gametophytes are induced on the callus growth and the gametophytes are transferred to said agar based novel culture mediumS Of course, if the agar based culture medium becomes depleted, the callus cells/gametocytes can be transferred to fresh medium The incubation is advantageously carried out at temperaturesof from 10 to 20 0C under a light intensity of from 1000 to 4000 1 x for a photoperiod of from 8 to 10 hours.

The preferred sterilant for said novel inoculum preferably is chloros at about 5 to 30% V/V most preferably at 10%V/V,the stipe of the seaweed being soaked therein for aDout 5 to 15 minutes, most preferably for 10 minutes.

The algal material is preferably obtained from healthy plants freshly collected from their natural environment and are best transported in sea water in suitable containers such on bags or bins to prevent disiccation.

The success of the inoculation step is largely attributable to the age and nature of algal material used, the method of surface sterilisation, and the form in which the material is inoculated. The age of the algal material can be defined in terms of the stipe length, which should be at least 10cm long, and most preferably from 10 to 20 cm long.

Advantageously the stipe material is surface sterilized and sections are selected from the region adjacent to the meristematic zone in preparation for inoculation on the agar medium of this invention to obtain callus growth. Suitably the sterilized material is inoculated as 0.25 to 0.75 cm high discs on to the agar based culture medium in sterile containers.

The above described two stage culture method of this invention, involving a preliminary plate culture to obtain callus growth with preferably development of gametophytes and a final suspension culture of the callus tissue or gametophytes has many advantages. The medium of this invention coupled with the careful selection of algal material results in an accelerated callus growth period (about 4 weeks) and rapid gametophyte formation (at about 6 weeks) whereas formerly the corresponding periods were measured in months. Additionally by obtaining viable gametophytes in this way, a healthy suspension culture can then be obtained which has a vast commercial potential in the opportunity now exists to develop mass growth under sterile environmentally stable conditions.Thus uniformity of biomass production could be maintained, and controlled much more easily than in the existing techniques of mariculturé mentioned hereinbefore.

So far, best results have been observed with members of the genus Laminaria but it is anticipated that the techniques have wider applications. In addition to the commercial potential of the suspension culture in terms of by-product exploitation, callus culture also has potential use in the fields of clone production and organogenesis of desirably seaweeds.

Further aspects of the invention include firstly the novel inoculum kit comprising seaweed cells in said novel agar based culture medium of the first aspect of the invention; and the novel suspension culture kit comprising seaweed cells suspended in a culture medium of the first aspect of the invention, but with the optional addition of phytohormones.

The present invention will now be further described by way of the following exemplary embodiment.

Example Health plants are collected from the open shore and transported in bins of sea water bags to prevent disiccation. Whole stipes are removed from the blades and the former soaked in 10% Chloros (a hypochlorite wash) for period of up to 10 m. Sterilized stipes are then washed three times in sterilized sea water. The surface layers are then scraped away using a sterile blade. Sections of stipe up to 1 mm deep are prepared from the region adjacent to the meristematic zone and up to half way down the organ. These are transferred on to sterile agar medium as shown in the Table for incubation. All procedures are completed in a sterile environment.

Incubation is carried ou in the normal way providing the following favoured conditions are maintained. Optimum growth is observed within a temperature range of from 10 to 200C, with exposure to a light intensity of 1000 to 4000 lx for from 8 to 16 hours.

Rapid formation of gametophyte stages occurs on the surface of the developing callus culture, within a period of 4 - 8 weeks. These can be identified with the naked eye by their dark brown, shiny appearance as compared with the callus. Under light microscopy x40 the gametophytes appear as heterotrichous (branching) filaments. These gametophyte filaments can be used directly in the production of suspension cultures.

The gametophyte stages which develop on the callus cultures can be grown on independently either in plate culture on the agar medium or into suspension culture using media shown in the Table above (excluding agar).

Suspension cultures are induced by removing gametophyte from the surface of callus, or directly from gametophyte plate culture, and transferring into sterile liquid culture. The resultant culture develops as a finely branching form which remains in suspension and grows on healthily under incubation conditions as outlined above.

This suspension culture of Laminaria is the final product of the invention, and has great potential for exploitation in mass culture techniques for uniform production of important constituents such as alginates, as discussed earlier.

Claims (18)

Claims

1. An algae culture medium comprising major inorganic salts1 minor inorganics salts and phytohormones made up in filter-sterilized sea water.

2. A algae culture medium according to claim 1 wherein the phytohormones are selected from 2,4 Dichlorophenoxyacetic, kinetin, and Indole acetic acid.

3. An algae culture medium according to any of claim 1 or 2 wherein the medium further comprises vitamins.

4. An algae culture medium according to claim 3 wherein the vitamins are selected from biotin, vitamin B12 and thiamine Hcl.

5. An algae culture medium according to any one of the preceding claims wherein the major elements are selected from NaN031, NaH2PO4. 2H2O, NH4cl, and Na2Sio3 .9H2O; and the minor elements comprise of trace elements being selected from CuSO4.5H2O,ZnSO4.7H2O, CoCl2.6H2O,Mncl2.4H2O, and Na2MoO4. 2H2O.

6. An algae culture medium according to any one of the preceding claims wherein the concentration of phytohormones is from 2.0 to 6.Omg per litre of filtersterilized sea water.

7. An algae culture medium according to claims on dependant on claim 3 wherein the concentration per litre of filter-sterilized sea water of the major inorganic salts, trace elements, ferric sequestrene, and vitamins are respectively from 100 to 200 mgl -1, from 0.15 to 0.25mgl11 from 0.25 to 0.75mgl-1 and from 0.05 to 0.15mgl'1

8. An algae culture medium according to any one of the preceding claims and further comprising agar or the like.

9. An inoculum kit for a sea weed suspension culture comprising viable seaweed cells obtained from a seaweed callus growth on an agar based culture medium as defined in claim 8.

10. A suspension seaweed culture kit, comprising seaweed cells suspended in a seaweed culture medium as defined in any one of claims 1 to 7.

11. A method of preparing an inoculum for a seaweed suspension culture comprising obtaining a seaweed stipe under sterile conditions; removing the surface layer of the stipe; inoculating under sterile conditions ,an agar based culture medium with cells from said sterilized stipe; and incubating the inoculated medium under conditions favourable for callus growth.

12. A method of preparing an inoculum according to claim 11 wherein the agar based culture medium is inoc-ulated with tissues selected from the meristermatic region or å region adjacent thereto of the sterilized stipe.

13. A method of preparing a seaweed suspension culture comprising preparing a seaweed culture medium according to anyone of claim 1 to 7 but with the phytohormones being an optional component; inoculating said prepared culture medium with seaweed cells obtained from a callus growth obtained according to claims 11 or 12; and then incubating the suspension culture under favourable growth conditions

14. A method of preparing a seaweed suspension culture according to claim 13 wherein the culture medium inoculated with cells from the callus growth, is as defined in any one of claims 1 to 7.

15. A method of preparing a seaweed suspension culture according to claim 13 or 14 wherein the inoculated suspension culture is suitable aerated.

16. A method of preparing an inoculum for a suspension culture according to claims 11 or 12, wherein the seaweed is the genus Laminaria of the division Phaeophyta.

17. - A seaweed culture medium substantially as described and as illustrated by the accompanying example.

18. A method of preparing an inoculum for seaweed suspension medium or a method of preparing seaweed suspension medium, substantially as described and as illustrated by the accompanying example.