This invention relates to a process for the storage of a culture or fungi. In particular, this invention relates to a process for the storage of Mycorrhizae, which belongs to class basidiomycetes of fungi.
Reports available on mycorrhizae describe that the fungi could not be subjected to the step of lyophilization or freeze drying because its sensitive cell structure. Most of the fungal where such a method of lyophilization has been applied are those which produce hard spores in synthetic medium. Mycorrhizae does not produce hard spores, and consequently could not be advantageously to the step of lyophilization. In fact, myrelia are delicate thread like structures, which are relatively short lived and highly sensitive. Due to such a delicate thread like structure, the organism dies when subjected to freeze-drying.
The known methods to store mycorrhizae had several disadvantages. One such disadvantage was the possibility of contamination. Another disadvantage was the loss of virulence. Yet another disadvantage was that during transportation the culture dies or gets contaminated. A further disadvantage is that of costs.

An object of this invention is to propose a process for the storage of mycorrhizae, which can be produced in dried form and packed in sealed ampoules.
Another object of this invention is to propose a process for the storage of mycorrhizae, which can avoid contamination.
Still another object of this invention is to propose a process for the storage of mycorrhizae, which no longer suffers from the disadvantage of the loss of virulence.
Yet another object of this invention is to propose a process for the storage of mycorrhizae, which has a ease of handling during transportation.
According to this invention there is provided a process for the storage of culture, such as
mycorrhizae, comprising in the steps of:
(i) selecting the fungus, namely Laccaria Laccata;
(it) introducing the fungus into a medium selected from potato dextrose agar or MMN
and having a pH of 7.0 to 7.5; (iii) allowing the fungus to age; (iv) subjecting the fungus to the step of pre-freezing in the presence of cryoprotectants
and such that the temperature of pre-freezing is maintained not above than -30°C, (v) subjecting the fungus to the step of freeze drying by a tube in tube method at a
temperature not above -100°C.

In accordance with this invention, the candidate fungus is first selected for the process of lyophihzation. Here the parameters considered were mainly the thickness of the vegetative mycelium and the growth rate of the fungal colony on solid MMN medium. The mycelium thickness was 0.2 to 1 micrometer. Such a thickness is chosen so that the material is sturdy. Further, the fungus should have a fast growth rate.
The fungus had a fast growth rate dependent on certain optimum conditions. The major
factors those affect growth are:
(i) Synthetic medium; two types of medium potato dextrose agar and MMN agar
were used for the growth of the fungus. Preferably, MMN agar is employed as the
medium, (ii) Temperature: As the fungi are temperature sensitive, the fungi was maintained at
25 °C constantly, (tit) Light: The fungi is root inhabiting and kept in dark during incubation, (iv) pH : The fungi exhibited a maximum growth of the fungal biomass by
maintaining the medium with a pH 7.0 to 7.5 and preferably 7.0. (v) Water activity: The organism requires water, which serves as a biological solvent
for all biochemical reactions. To obtain a good growth of the fungus, the % agar
in the medium was varied from 0.6 to 1.2% which directly affects the water
availability to the fungus in the synthetics medium. Preferably 0.8% agar is
present in the medium.

(vi) Aeration: As these are aerobic fungi, solid agar grown culture was suitable for proceeding to lyophilisation.
Fungi of age above three weeks and below two months of age are suitable for freeze-drying by the process of the present invention.
Tn accordance with this invention, the process comprises a two step process of
lyophilization consisting of a gradual pre-freezing step followed by freeze-drying. The
step of lyophilization is carried out in the presence of cryoprotectants. By way of
example and with implying any limitation, the cryoprotectants with their concentration
are selected from.
Glycerol 5%, 10%, 15% 20% (with and without 1% inositol),
DMSO 5%, 10%, 15%, 20% (with and without 1% inositol),
Sucrose 10%, 20%
Methanol 5%, 10%
Ringers solution,
PBS,
BSS,
PVP,
Trehalose 0.1M, 0.2M and
Sterile water as control.

The cryoprotectants are sterilized by any known method. The culture with the cryoprotectants is placed in a cooling bath, such as containing solvent methanol, at room temperature. The temperature of the bath is gradually brought down to -30°C at a rate of approximately 2°C/min. with constant manual stirring to prevent any temperature localization effect. Once, the bath temperature reaches at least -30°C, it is maintained constant and the mycelial discs are allowed to freeze for a period of for example 1 and l/t hours. Then the final withdrawal is made. Preferably, 10% DMSO is taken as the most suitable protectant.
Reference is made to the step of pre-freezing described hereinabove. It was found that when the culture was kept in an evacuating chamber and subjected to the subsequent step of freeze drying, then appeared a liquification of the cryoprotectant in the vials. It is believed that the vials maintained at 30°C were suddenly being exposed to the chamber temperature which would be initially equal to the room temperature. This temperature gradient of around 60°C (-30°C to + 30°C (room temperature)) which exists till the chamber cools down to -30°C, would cause liquification of the frozen solute surrounding the mycelial cultures disturbing the chemical equilibrium of the cell and surroundings. Therefore, the present invention minimizes the temperature gradient as effectively as possible. For this purpose, the process comprises a tube in tube method. To crushed ice, sodium chloride and calcium chloride were added and thoroughly mixed. This ice

mixture is added to long lyophilizer tubes. The tubes containing the fungi are taken out from the prefreezing bath and immediately plunged into the long lyophilizer tubes with crushed ice mixture inside. The mouth of the inner tube is sealed with parafilm leaving a small aperture to facilitate escape of moisture. The long tubes are then loaded on to the posts provided on the freeze-drying equipment. The drying was carried out for a period of 10 hours and once the ran is complete, the tubes are unloaded and the mycelial discs are immediately reconstituted.
Reference is now made to the step of freeze-drying. Firstly, the condenser temperature should be always lower than the product temperature to allow migration of solid water away from the product.
To achieve this, the condenser should be cooled long enough, before loading the samples for freeze-drying. Secondly, the freeze-drying module should provide an environment that allows water molecules to migrate from the frozen product This has been achieved in our process by evacuating the chamber prior to loading the samples. The temperature of the condenser is brought down to -70°C or below and the freeze-drying chamber evacuated to a pressure below 125 units prior loading the samples. During the process of freeze drying, the temperature goes down to -100°C ± 2°C, and the final pressure is below 100 units. When once the pre-freezing is completed with the holding temperature, the tubes are transferred to long lyophilizer tubes (tube in tube method) with crushed ice mixture and immediately loaded on to the freeze-drying equipment.

Once the pre-freezing was optimized and measures were taken to overcome the temperature gradient during the transition from pre-freezing to freeze-drying, the next step is the extent of freeze-drying. The freeze-drying protocol used shows a freeze-drying run of 6-9 hrs. said period being dependent on parameters such as the measurements of the inner and outer tubes, amount of culture and protectant, and the amount of surrounding crushed ice mixture.
In order to obtain a viable culture from the freeze-dried product, the dried culture needs to be reconstituted. Reconstitution of a freeze-dried specimen mainly requires rehydration. The active sites in a freeze-dried product are exposed because of the large surface area of the dried matrix. Due to the removal of the solvent from the cells, there is a higher concentration of intracellular solutes. Thus, when rehydrating the cells, care is taken to maintain the delicate balance of the cellular chemistry of cells.
The major factors affecting the process of reconstitution are:
(i) type of rehydrant used while reconstituting the cells,
(ii) extent of rehydration,
(tit) temperature of rehydrant
(iv) growth conditions provided to the freeze-dried specimen after reconstitution.

The rehydrating agents employed upon the freeze-dried material are :
Sterile water,
Sterile water + peptone (0.1%, 0.5%, 1%).
The length of rehydration time optimum for the freeze-dried test fungus, rehydration was
carried out for extended periods 1 to 12 hours. The rehydrated mycelial discs were placed
on fresh MMN agar plates, and incubated to observe the growth of the culture.
EXAMPLE
For a culture vial with three discs of freeze-dried mycelium inoculam, 1 ml of sterile water to be added and allowed to rehydrate for a minute. Then 1 ml of 1% peptone (previously sterilized) is to be added. The culture discs need to be kept in the solvent for a period of 12 hours to rehydrate. The rehydrated culture discs are to be placed on a fresh MMN agar plate. The plates containing desired on MMN agar are to be incubated in dark at a temperature of 25 °C. The appearance of mycelium filaments growing from the perimeter of the disc denote a viable culture.
The above protocol has resulted in complete viability of freeze-dried Laccaria cultures.

WE CLAIM:
1. A process for the storage of culture, such as mycorrhizae, comprising in the steps
of:
(i) selecting the fungus, namely Laccaria Laceata;
(ii) introducing the fungus into a medium selected from potato dextrose agar or MMN
and having a pH of 7.0 to 7.5; (iii) allowing the fungus to age; (iv) subjecting the fungus to the step of pre-freezing in the presence of cryoprotectants
and such that the temperature of pre-freezing is maintained not above than -30°C, (v) subjecting the fungus to the step of freeze drying by a tube in tube method at a
temperature not above -100°C.
2. A process as claimed in claim 1 wherein the fungus is maintained at 25 °C in said medium and under dark conditions.
3. A process as claimed in claim 1 wherein the agar is present in the medium to the extent of 0.6 to 1.2%.
4. A process as claimed in claim 1 wherein the fungi has an age of 3 weeks to 2 months.

5. A process as claimed in claim 1 wherein the cryoproteetants are selected from glycerol, BMSG, sucrose, methanol, ringers solution, PBS, BSS, PVP, trehalose and sterile water.
6. A process as claimed in claim 1 wherein the step of pre-freezing comprises in reducing the temperature of a pre-freezing or cooling bath to at least -30°C at a rate of approximately 2°C/min.
7. A process as claimed in claim 6 wherein said temperature of the bath with the fiingi is maintained for a period of 1 1/2 hours.
8. A process as claimed in claim 1 wherein the step of freeze-drying consists in maintaining the condenser temperature lower than the fungi temperature.
9. A process as claimed in claim 8 wherein the chamber is evacuated prior to loading with the fungi.
10. A process as claimed in claims 8 and 9 wherein the temperature is reduced to -100°C±2°C and a pressure below 100 units.

11. A process as claimed in claims 8 to 10 wherein the step of freeze drying is carried out for a period of 6 to 9 hours.
12. A process for the storage of culture such as mycorrhizae substantially as herein described.
13. A process for revitalizing freeze dried mycorrhizae which comprises in reconstituting the dried culture in a rehydrant for a period to reconstitute the culture, placing the disc containing the reconstituted culture on a MMN agar plate and subjecting it to the step of incubation.
14. A process as claimed in claim 13 wherein the step of incubation is carried out in darkness and at a temperature of 25°C.
15. A process as claimed in claim 13 wherein the solvent comprises sterile water and peptone.
16. A process as claimed in claim 13 wherein the culture discs are kept in the solvent for 1 to 12 hours.
17. A process for revitalizing freeze-dried mycorrhizae substantially as herein described.