India is primarily an agriculture-based country. It offers vast potential for the cultivation of mushrooms due to the presence of abundant raw material and conductive climate for its growth. Cultivating mushrooms is an ideal income generating activity for small scale and, landless farmers which besides generating income also supplements nutrition.
LIFE CYCLE OF MUSHROOM
Mushrooms are fungi, which do not contain chlorophyll. They are saprophytes and obtain their food by colonizing dead organic matter. In nature, mushrooms multiply by producing millions and millions of spores. When spores are fallen in a suitable environment, they germinate and branch to form a mycelium. The vegetative body is made up of filamentous, tubular structures called hyphae. The mass of hyphae is termed as mycelium. When the mycelium derives enough food from the growing substratum and under favorable conditions again the sporocarps (fruit bodies/ mushrooms) are produced.
Mushrooms produce seeds or spores under the cap on gills which are ridge-like structures. These spores are main source of mushroom propagation but cannot be used for mushroom cultivation because they are very small in size and difficult to handle. Moreover, these spores are very slow growing and may not give mushroom fruit bodies. Therefore, vegetative mycelium of mushroom species is grown on cereal grains and used as seed.
MUSHROOM SPAWN PRODUCTION
Spawn is the seed material used for mushroom cultivation. It is the mycelium of the mushroom grown in a suitable supporting medium. Spawn is the basic material which decides the success or failure on the production of a particular mushroom. The entire process of mushroom spawn production is carried out in scientific laboratory. The steps involved in spawn production are similar for all the commercially cultivated mushrooms. The yield and quality of spawn is governed by the genetic makeup of the strain used, quality of substrate used and the absence of any contaminants.
Establishment of a pure culture of the desired species is the first step for spawn production. A pure culture is to be obtained by tissue culturing of the desired mushroom in a culture medium. Potato Dextrose Agar (PDA) is usually used as culture medium. After preparing a sterile culture medium, a small portion of the tissue of the desired mushroom is planted on the medium in aseptic conditions and incubated. By two or three frequent transfers to fresh sterile medium slants (sub culturing), the pure culture is obtained. This pure culture can be used as stock culture, which can be used for preparation of spawn. The isolation and culturing of mushroom tissue requires technical expertise and highly aseptic conditions.
PREPARATION OF POTATO DEXTROSE AGAR (PDA) MEDIUM
Materials required for preparation of 1 litre PDA medium
Potato- 200g
Dextrose- 20g
Agar agar- 20g
Distilled water- 1 litre
Test tubes- 100 nos.
Glass funnel with mesh- one
Pressure cooker/autoclave
Non absorbent cotton roll- one
Conical flask (1 litre)- 3 nos.
Procedure:
Peel off the skin of potatoes and cut them into small pieces. Cook the potatoes in 400 ml distilled water for 1 hour or steam for 30 minutes in one conical flask in a pressure cooker. Melt 20g agar agar in another 400 ml of distilled water, heating over a water bath, in one conical flask. Strain the cooked potatoes through a clean cloth piece. Add glucose to the filtered potato extract. Pour melted agar in the potato extract while hot in to the third conical flask. Thoroughly mix it and make up the volume to 1 litre. Pour 10-15 ml. of the PDA medium in each test tube. Plug the mouth of test tube with non - absorbent cotton. Sterilize the medium filled in the test tubes in an autoclave / pressure cooker for 20 minutes (at 1.05 kg /cm2 and 121o C) after hearing the first whistle. After sterilization, keep the test tubes in a slanting position by providing a suitable support on a plain table so as to solidify the medium into slants. The medium will be solidified within half an hour. The prepared slants are used for raising culture. Check the test tube in stock to observe any contamination. If any contaminated test tube is observed, they are discarded. These slants after cooling can be used for culturing purpose. Avoid using very old and dried PDA medium.
PREPARATION OF PURE CULTURE
Tissue culture
Select high yielding, freshly harvested, early producing mushroom at the button stage of development for preparation of spawn. The selected mushroom is split/cut in to two half with the help of knife/blade length wise. With a sterilised needle, a tiny bit of tissue is picked up from the newly exposed surface, and is inserted into agar slants. Portion where the gill plate joins the stipe is considered the best tissue for excision. The inoculated slants are incubated at 24-26oC preferably in darkness for about 10-15 days. All the operations should be done aseptically over a flame. Inoculation must be done under hygienic conditions in laminar air flow chamber or in inoculation hood.
SUB CULTURING
Sub culturing of the tissue culture slants are done to maintain and multiply the cultures. For sub culturing, a small bit of the mycelium along with the medium of the pure culture is taken with the help of a culture rod and put it on another sterile PDA slant.
PRESERVATION AND STORAGE OF CULTURE
Proper maintenance of pure cultures/stock cultures of mushroom is necessary to maintain vigor and productivity. Mushroom spawn prepared by inoculating the pure culture of the desired mushroom species after its full growth is called ‘mother spawn’.
PREPARATION OF SUBSTRATE FOR SPAWN MULTIPLICATION:
Graminaceous grains (namely paddy, wheat, sorghum, maize etc.) are used as substrate for preparation of spawn. It is desirable that the grains has not been treated with insecticides or fungicides.
Procedure (using paddy grains)
The healthy grains are soaked in calcium carbonate overnight. The grains selected should not be insect damaged. The grains are thoroughly washed 2 to 3 times in water to remove solid debris, straw materials or undesirable weed seeds. Grains are washedand then boiled (half cook the grains) for 20-30 minutes. The grain should not split and release starch. Over cooking and under cooking of the grains may lead to contamination and spoilage of the spawn. The excess water is drained off and the grains are mixed with calcium carbonate (@ 30 - 50g/kg of dry grains used) such that each grain gets a white powdery coating, to adjust the pH of the grains to 7 and also to check the sticking of the grains. The grains are then filled into glass drip bottles/ Poly Propylene (PP) covers (250 -300g). Bottles/ PP cover are plugged with non-absorbent cotton/stapled and sterilized (at 1.05kg/cm2 121oC) for 1½ to 2 hours. Sterilized bottles/ packets are taken out from the autoclave and allowed to cool. After cooling, sterilized substrates in bottles/PP covers are kept in inoculation chamber/ laminar airflow chamber. Inoculation is done under aseptic conditions with required mother/ stock culture of the desired mushroom. Platform of the laminar flow chamber/inoculation hood should be cleaned either with spirit/dettol dipped in cotton. With the help of a culture rod, a small quantity of pure culture of the desired mushroom is transferred to the sterilized containers (previously kept ready) aseptically.
Label the containers with the name of species and date of inoculation. The inoculated bottles / packets are incubated in a convenient place in shelves and observe for mycelial growth. The white color mycelium will normally grow from the pure inoculated from the top and will spread to bottom slowly.
Observation should be made to check for any contaminant growth inside. The mycelium will be harbored fully in the substrate in the container by about 15-18 days time.
MULTIPLICATION OF BED SPAWN FROM MOTHER SPAWN
The fully-grown spawn can be multiplied by transferring in fresh sterile substrate containing bottles/packets in aseptic conditions. One bottle of mother spawn is sufficient to multiply in 20-30 bottles/polypropylene bags for the purpose of ‘bed spawn’.
By this method the spawn will be ready by 12-15 days time for the preparation of beds. Shake the inoculated bottles thoroughly and incubate them at 24 -26o C for the growth of mycelium.
Any discoloration such as greenish or blackish color in the spawn is an indication of contamination. The inoculated bottles are checked daily for any contamination and such ones are removed regularly from the lot.
STORAGE OF SPAWN
Fresh spawn is always preferred for cultivation purpose. Healthy spawns can be stored for a maximum period of 6 months at 4oC. When the refrigerated spawn is used for cultivation, it should be allowed to attain room temperature before its use.
TRANSIT OF SPAWN
During transportation, spawn should not be exposed to temperatures higher than 35oC as higher temperature is detrimental to mushroom mycelium. Spawn bags/bottles should be taken out of packing and kept on convenient place in shelves/ racks. Once the spawn bottle/packet is opened, that should be used entirely.
EQUIPMENTS AND OTHER MATERIALS REQUIRED
FOR A HOMESTEAD SPAWN LABORATORY
1.Pressure Cookers
2.Boiling vessels
3.Inoculation hood/laminar air flow
4.Refrigerator
5.Glassware
6.Chemicals
7.Non-absorbent cotton
8.Polypropylene bags or bottles
9.Steel racks
10.Exhaust fans
11.Gas connection with burner
12.Culture rod (Inoculation needle)
13.Forceps
14.Ethyl alcohol/rectified spirit
15.Culture rod
16.Knife/blade
John Jo Varghese
Subject Matter Specialist (Agronomy)
Mitraniketan Krishi Vigyan Kendra
Vellanad
Thiruvananthapuram
Kerala, India 695 543
Ph: 0472-2882086
Email: trivandrumkvk@yahoo.co.in
Web: www.mitrakvk.org
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