Spawn production technology

(Seed for mushroom)

 Contents
Spawn production in entrepreneurs view
Basic life cycle of mushroom
Spawn production methodology
Quality standards for mushroom spawn
Setting up a lab
Economics of spawn production
Services (spawn) from ICAR-DMR
Practical exposure
• Do I need an exclusive spawn lab for the
proposed/existing mushroom production unit ?

• If I start a separate spawn laboratory, What will be

its future ?
 Mushroom production in India

Agaricus Pleurotus Volvariella Calocybe Lentinula Total

94,676 21,271 9,553 3,179 1,103 1,29,782
State Button Oyster Milky Others Total
AP 3000 500 15 0 3515
Delhi 3000 50 20 0 3070
Goa 4200 20 0 0 4220
Gujarat 10000 1200 0 0 11200
Haryana 15000 50 50 0 15100
HP 9000 110 30 10 9150
Maharashtra 10000 2000 50 0 12050
Odisha 126 6310 0 9550 15986
Punjab 16000 2000 0 0 18000
TN 6500 2000 1500 0 10000
UK 8189 1228 819 0 10236
UP 7000 100 0 0 7100
Grand total 119627
Basic life cycle of mushrooms

Myco vocabulary
 Steps in spawn production

Raising of pure culture

Preparation of master spawn

Multiplication of master spawn

Raising pure culture
 Steps in preparation of pure culture

Preparation of culture medium

Isolation of tissue from healthy fruit body
Inoculation
Incubation
Sub culturing and storage
 Possible errors
• Absence of mycelial growth from the tissue after 3-4 days of
incubation
– Due to use of over heated inoculation needle
– Keeping the tissue in front of flame for long duration
– Non suitability of medium
– Non suitability of culture medium pH
• Contamination
– Coloured mycelial growth
– Slimy or shining growth
 S. Type of Main Agar- pH Method of preparation
No medium ingredient Agar
1 Potato Peeled and 20 g 7.0- Potato pieces/slices are boiled in water for 15-
Dextrose sliced potato 7.5 20 minutes till they become soft. Water is
Agar (PDA) (250 g) + strained to remove the sliced potato pieces and
Dextrose (20 collect the decoction/filtrate. Volume of filtrate
g) is made to 1 litre by adding water.
2 Malt Extract Malt extract 20 g 7.0- Add 20 g of malt extract powder and 20 g of
Agar (MEA) (20 g) 7.5 agar agar powder to 1 litre of water and boil it
thoroughly.
3 Compost Pasteurised 20 g 7.0- Compost is boiled in 2 litres water for 2 hours
Extract Agar compost (200 7.5 or till half of the water is evaporated, strained
(CEA) g) after 24 hours and the volume of supernatant is
made to 1 litre with water, agar is added and
medium is sterilized.
4 Wheat Wheat grains 20 g 7.0- Wheat grains are boiled in water for 2 hours,
Extract Agar (32 g) 7.5 strained after 24 hours and the volume of
(WEA) supernatant is made to 1 litre with water, agar is
added to the supernatant and then sterilized.
 Preparation of master spawn
• Pure culture raised from tissue will be inoculated in a
suitable substrate
• Wheat, Jowar, Bajra, Rye, paddy etc. which provides food to
the growing mycelium
• Grains must be clean, unbroken, and free from any infection
and of uniform size
 Preparation of master spawn

I • Boil the grains in water

II • Remove excess water through sieve and dry the grains

III • Mix Gypsum and lime (0.5% and 2%)

IV • Fill 300g of substrate in glass bottles

V • Plug non absorbent cotton and sterilize at 22 p.s.i pressure

VI • Inoculation

VII • Incubation
 Large scale multiplication of master spawn

I • Boil the grains in water

II • Remove excess water through sieve and dry the grains

III • Mix Gypsum and lime (0.5% and 2%)

IV • Fill 1 kg of substrate in heat resistant polypropylene bags

V • Plug non absorbent cotton and sterilize at 22 p.s.i pressure

VI • Inoculation

VII • Incubation
 Some necessary Do’s
• Over-boiling and under-boiling of wheat grains should be
avoided
• Sterilization should be carried out at strictly recommended
pressure and for the recommended period
• Strict hygiene should be followed
• Bottles should be shaken at an interval of 4-5 days to trigger
the growth.
• Before the use of master culture for inoculation in
commercial spawn bags, they must be checked for any sort of
contamination.
 How to judge the quality
 Spawn prepared on jowar or wheat grains gives higher yield
over spawn prepared on bajra, barley or paddy grains
 There should be proper coating of mycelium around grains
used as substrate for spawn production
 The growth of fresh spawn is more or less white
 There should not be any slimy growth in spawn bags/bottles
 There should not be any greenish or blackish spot in the
spawn bottles/bags
 When the spawn bags/bottles are opened for spawning it
should emit typical mushroom smell
Quality standards for mushroom spawn

Nucleus seed

Breeders seed

Foundation seed

Truthfully labelled seed

 Breeders seed Foundation seed TL seed
Pure culture Mother spawn Commercial spawn
Should possess the Second class fungal with Spawn should always be
original morphology white hyphae, strong and prepared from master
characteristics dense spawn
should be obtained from Master Spawn should It should be multiplied on
Research organization or always be from pure wheat, jowar, bajra or
authentic source culture barley grains
Free from contamination Free from contamination Free from contamination
Culture should indicate Should be produced in Should be produced in
specific growth rate on autoclavable transparent autoclavable
defined medium and at glass bottles polypropylene bags
defined temperature
 Breeders seed / pure culture
Character Button Oyster Straw Milky Shiitake
mushroom
Medium CEA WEA PEA WEA / PEA SEA
Morphology Off white in Pure white Cottony Pure white, Initially
colour and thick fluffy with dense and pure white,
with fluffy brown thick with then turning
growth sclerotia fluffy to light
growth brown
Storage 18-22°C 18-22°C 18-22°C 18-22°C 18-22°C
temperature
Frequency Once in Once in Once in Once in Once in
of sub every 2 every 2 every 2 every 2 every 2
culturing months months months months months
 Foundation seed / Mother spawn
Character Button Oyster Straw Milky Shiitake
mushroom
Incubation 25±2°C 25±2°C 32±2°C 32±2°C 25±2°C
temperature
Storage 4-6°C 4-6°C 18-20°C 18-20°C 4-6°C
temperature maximum maximum
for 30-40 for 30-40
days days
Age of should not should not should not should not should not
mother be older be older be older be older be older
spawn than 60 than 30 than 30 than 30 than 30
days days days days days
 Certified seed / commercial spawn
Character Button Oyster Straw Milky Shiitake
mushroom
Incubation 25±2°C 25±2°C 32±2°C 32±2°C 25±2°C
temperature
Storage 4-6°C 4-6°C 18-20°C 18-20°C 4-6°C
temperature maximum maximum
for 30-40 for 30-40
days days
Age of should not should not should not should not should not
mother be older be older be older be older be older
spawn than 60 than 40 than 30-40 than 30-40 than 45-60
days days days days days
 Design of a spawn lab
Safety
Comfort
Energy efficiency

Positive pressure area

 Positive pressure air flow

Air will flow out of the room instead of in

 Basic equipments needed for spawn lab
Equipment Purpose
Boiling For boiling the grains.
pans/boiling vessel
Autoclave For dry heat sterilization of wheat grains.
Laminar flowFor creating the aseptic conditions for isolation and multiplication of
cabinet cultures.
BOD incubator For creating the favourable conditions for multiplication of vegetative
cultures and the master spawn.
Refrigerator For short-term preservation of mycelial cultures.
Electronic Balance For accurate weighing of components for medium preparation, spawn
production
pH meter For determining the pH and its maintenance.
AC systems For maintaining desired temperature in the incubation room
Generator For uninterrupted electricity supply which is essential for proper
functioning of spawn laboratory.
Miscellaneous Exhaust fans, filters, working tables, troughs, sieves, inoculating
needles, scalpels, test tubes, petri plates, etc. are required for different
operations in spawn preparation.
 High pressure sterilization
Pre conditioning --- Exposure ---- Post conditioning
• Time to reach equivalent microbial lethality
• The temperature of saturated steam is directly related to the
pressure at which it is controlled. A typical cycle at 121 C requires
15 to 17 lbs of gauge pressure in the chamber
• Super heated steam that is above its saturation temperature will
denature the protein
• Direct steam contact with the surface of the object to be sterilized
is required for the steam to transfer its stored energy to the object.
• Air removal is required to avoid the vacuum leaks and poor steam
quality
• Wrapped items must be dry before they can be aseptically removed
from the sterilizer.
Laminar air flow cabinet
 FAQs
 (How to prevent the contamination in bags)n
 What is the frequency of sub culturing
 We are unable to maintain the culture of button mushroom in
our lab
 What is the moisture level to be maintained in the millet
 What is the ratio of gypsum and lime to be added
 How to prevent air borne contamination
 Is there any chances of using UV lights in incubation room for
disinfection
 Do I need any certification to establish a spawn laboratory
 Where to find the buyers for my spawn
Thiophanate methyl @ 0.05 g/ kg Streptomycin / neomycin @ 10
of boiled grains mg/ kg of boiled grains
(Alban / Fungo)
 Services offered by ICAR-DMR

• Sale of Culture, mother spawn and commercial

spawn
• Capacity building through hands on training
programmes
• Preparation of TEFR for establishing the new spawn
lab
 List of commercial strains
Mushroom species Strain number Specific character
White button mushroom NBS-5 Non browning strain
U3
Agaricus bitroquis DMR-AB-5 High temperature tolerant
Brown button mushroom DMR-Button-06 Brown in color
Oyster mushroom DMRP-112
DMRP-205 Pink oyster
DMRP-88
DMRP-136 Grey oyster
DMRP-135 King oyster
Milky mushroom DMR-Milky- 334 & 335
Shiitake mushroom DMR-Shiitake – 356
DMR-Shiitake – 34
Paddy straw mushroom DMRO-247, 484