Mushroom Spawn (Seed) Production/Procurement

1 19 Mushroom Spawn (Seed) Production/ProcurementMushroom ProductionNotes2 Mushroom Spawn (SEED)PRODUCTION/PROCUREMENTIn the first lesson we have learnt about different types of Mushroom and basic stepsinvolved in their cultivation. We came to know that seed of Mushroom is calledspawn and producing or procuring good quality seed of Mushroom is the first stepin cultivation of Mushroom . Many big commercial units make their own , small scale growers normally purchase it. While purchasing, transportingand storing seed of any crop, we take precautions like selecting the correct variety,checking the quality and transport and store it in a way that its quality is notaffected. Same is true for Mushroom seed. We will discuss about these aspects inthe current reading this lesson you will be able to:zdescribe the steps involved in preparation of grain Spawn ;zidentify the equipments required in Spawn lab;ztake precautions during purchase, transport and storage of Spawn PREPARATIONB efore starting Spawn production, we can prepare or procure the pure culture ofmushroom.

2 Culture of Mushroom is the fungus obtained from the Mushroom fruitbody under sterile conditions. We have equipment Laminar Flow/Clean air stationthat gives sterile air. You will see further that we use autoclaves to sterilize , the Spawn is produced in the lab and cannot be made in open like in othercrops. 20 NotesMushroom Spawn (Seed) Production/ProcurementMushroom ProductionFig. : Taking Mushroom from pileus and stipejunction for tissue cultureFig. : Growth of the part of fruit-body into myceliumafter transfer to test tubes on mediaTo prepare the pure culture we take a Mushroom (Fig. ) or its spores and allowgrowing it artificially in a tube containing the culture media (Fig. ). The tubeshave media like Potato dextrose agar, malt extract agar, etc. The media is made freefrom living organisms ( sterilized) and all the work of preparing cultures is doneunder sterile conditions.

3 For preparation of Spawn , the fungus from the culture isthen grown on grains or any other suitable medium. 21 Mushroom Spawn (Seed) Production/ProcurementMushroom ProductionNoteszCereal grains like wheat, sorghum, pearl millet, etc can be used for makingspawn. Wheat grains are the most commonly used substrate. The wheat grainsare boiled partly or half cooked after soaking overnight so that the grainsbecome slightly soft but do not burst. After this grains are kept on a sieve todrain out the excess water and are allowed to dry for evaporation of surfacewater (Fig. ).Fig. : Boiled grains on sievezNext step is to add chalk/calcium carbonate ( ) and gypsum (2%) mixthoroughly (Fig. ) and fill in bottles/ polypropylene bags. However, nowa day s only calcium carbonate is enough to mix in the grains and its mixingmaintains the desired : Grains mixed with chalk & gypsumzThe bottles are plugged using plugs made of non-absorbent cotton.

4 Thesedays most of the growers use polyfill (a synthetic material which you musthave felt in soft pillows) instead of cotton as it is cheaper and gives betterresults (Fig. ).Fig. : Plugging of bottles/ bags 22 NotesMushroom Spawn (Seed) Production/ProcurementMushroom ProductionzThese filled bottles/bags are sterilsed in special equipment called can be imagined as a big pressure cooker. The bottles/bags areautoclaved for hour at pressure of 22 pounds per square inch (psi). Thetemperature inside the autoclave reaches to about C. There is pressuregauge attached to the autoclave and you can directly read the autoclaving is complete and it has cooled down, the bottles/bags aretaken out, cooled and inoculated with Mushroom culture and this operationis done in laminar flow (an equipment that has filters so that only sterile aircomes out) or under totally sterile conditions (Fig.)

5 Fig. : InoculationzAfter inoculation, the bottles are incubated (kept) at 25 C for 2-3 weeks whenthe Mushroom mycelium fully colonizes (covers) the wheat grains (Fig. ).To begin with, Spawn is prepared in bottles using the culture grown in the testtube and this is called Mother Spawn . The first generation grain spawnprepared in glass bottles is Mother : Mother spawnzMother Spawn is used to make commercial Spawn which is prepared inpolypropylene (PP) bags (Fig. ). That is, few grains of readymade spawnare added in each bag (Fig. ). It is not recommended to multiply Spawn tospawn beyond two generations. Polypropylene bags are used as these can be 23 Mushroom Spawn (Seed) Production/ProcurementMushroom ProductionNotesautoclaved and are cheap and easy to transport (normal plastic bags will melt).

6 These are then incubated for 3 weeks (Fig. ). In between we inspect thesebags, shake once or twice and remove bags that might have gone : PP bagsFig. : Spawn to spawnFig. : IncubationNormally it takes 15-20 days for complete spread of mycelium on the grains. Majorsteps in preparation of Spawn have been shown in Fig. 24 NotesMushroom Spawn (Seed) Production/ProcurementMushroom ProductionFig. : Major steps in Spawn multiplicationSome of the important precautions while preparing Spawn keep the inoculation chamber (area where we prepare culture ortransfer culture from tube to bottles or from bottle to bags) and its surroundingsvery on UV tube in the inoculation chamber for 30 minutes beforeinoculation by keeping sterilized substrate, forceps, etc. inside the off UV tube 15 minutes before you enter the inoculation should always be done near the spirit lamp flame to should swab your hands and clean inoculation area using Spawn should not be used beyond two generations as it starts todegenerate.

7 Fresh Spawn gives higher yield; therefore, Spawn may not bestored for more than a bottles and bags after 10 days or so to ensure uniform white silkygrowth and also to remove contaminated bottle (that is bottles in which wesee green, black or other unwanted growth which is due to entry of spores ofother fungi despite our precautions). the bottles must be labelled indicating firms name, species, variety, dateof inoculation to know the age and type of Spawn . 25 Mushroom Spawn (Seed) Production/ProcurementMushroom ProductionNotesINTEXT QUESTIONS True or False(i) Mother Spawn is the Spawn produced from pure culture in test tubes.(ii) Poly-fill can be used instead of non-absorbent cotton for plugging the bottles.(iii)Pure culture can be prepared either from fruit-body of Mushroom or itsspores.

8 (iv)Mother Spawn should not be used for Spawn to Spawn multiplication beyond2-3 generations.(v) When the pressure in autoclave is 22 lb psi, the temperature inside EQUIPMENTS REQUIRED IN Spawn LABD ifferent equipments are required in a Spawn laboratory (Fig. ). pans/boiling kettle (vessel) for boiling the grains. We may boil grainin big utensils or boil in steam operated kettle if baby boiler is kettles working on electricity, kerosene or gas can be stove or steam line for boiling of wheat grains and preparation of digital pH meter to check pH of the for sterilization of Spawn /medium and oven for sterilization ofglassware. Two electrically operated autoclaves with 100-145 bottle capacityhaving a diameter of 2 and height of 3 are sufficient. If boiler is available,steam operated autoclaves can be used for better efficiency.

9 A small clinicalautoclave can also be kept for sterilization of culture (Biological Oxygen Demand) incubator is needed to incubate flow cabinet (normally 4 ft. horizontal) is needed for isolation andmultiplication of cultures and is needed for short-term preservation of mycelial items like glassware, chemicals for medium preparation, non-absorbentcotton, polypropylene bags (or bottles), disinfectant (formaldehyde), calciumcarbonate, calcium sulphate are also required. 26 NotesMushroom Spawn (Seed) Production/ProcurementMushroom racks in incubation room and cold storage for keeping bags/ bottles,exhaust fans, filters, office table, working tables, troughs, sieves, inoculatingneedles, scalpels, test tubes, Petri plates etc. are also Air Handling Units (AHUs) fitted with HEPA filters can be installed forcreating positive pressure in Spawn lab, particularly in inoculation , air curtains are desirable to keep aseptic conditions in the kettleAutoclaveLaminar flowBOD IncubatorFig.

10 : Equipments required in Spawn labINTEXT QUESTIONS True or False(i) A pH meter is used to sterilise the grains.(ii) BOD incubator is used for getting sterile air.(iii)Laminar Flow Cabinet is used to store cultures.(iv)Baby boiler is must for boiling the grains. 27 Mushroom Spawn (Seed) Production/ProcurementMushroom Spawn STORAGEF ully colonized bags, that is, the bags in which mycelium has covered all the grainsand Spawn is ready to use, can be stored in cold room (+4-6 C) for 1-2 months forfuture use. The Spawn of button Mushroom , Pleurotus can be stored at thistemperature. However, neither the culture nor Spawn of Volvariella, Ganodermaand Calocybe is stored below 15 C (Table ). The contaminated bottles/ bags/tubes etc. are autoclaved before emptying and discarding.