Biofertilizers:

Agricultural biotechnology:
• Biofertilizers:
• Biofertilizers are defined as biologically active products or microbial inoculants of
bacteria, algae and fungi, which may help biological nitrogen fixation,
solubilization of soil phosphorous, sulfur etc for the benefit of plants
• Soil is the natural habitat of variety of agriculturally beneficial microorganisms.
Certain soil microorganisms have ability to absorb and convert atmospheric
nitrogen to readily available form to the plants. Where as certain soil
microorganisms solubilize part of the bound phosphate of the soil and make
them available to the plants. Both these attribute make them important to be
used as biofertilizers
• Biofertilizers also include organic fertilizers which are rendered in available form
due to the interaction of microorganisms or due to association of plants
Biofertilizers include the followings:
1. Symbiotic nitrogen fixer- Rhizobium spp.
2. Asymbiotic free nitrogen fixer- Azotobacter, Azospirillum
3. Algae biofertilizer- blue green algae or its association with Azolla
4. Mycorrhiza
5. Organic fertilizers
Benefits of using biofertilizers:
• Increase crop yield by 20-30%
• Replace chemical fertilizers by 25%
• Stimulate plant growth
• Activate the soil biologically
• Restore natural soil fertility
• Provides protection against drought and some soil borne diseases
• Economic and ecofriendly
Nitrogen fixation:
• Atmospheric nitrogen is converted into combine form of organic compounds by
some microorganisms through biological reactions and the phenomenon is
known as biological nitrogen fixation or Diazotrophy
• There are two types of biological nitrogen fixation:
1. Non-symbiotic nitrogen fixation
2. Symbiotic nitrogen fixation
Non-symbiotic nitrogen fixation:
• It is brought about by the free living soil diazotrophs such as Azonomas,
Azobacter,, Bacillus, Clostridium, Aulosira etc
Symbiotic nitrogen fixation:
• Some microorganisms which develop symbiotic relationship with different parts
of plants and may develop special structures as the site of nitrogen fixation, for
examples- Rhizobium, Cyanobacteria, Nostoc etc
• It is the free living GM–ve , non- sporulating, aerobic, motile bacterium, which
reside in soil, capable of forming nodule, establishing symbiosis and N2 fixation
• Rhizobia are more prominent in the rhizosphere of leguminous plants.
• Examples are: R legumicosarium, R trifoli, R japonicum, R lupine
• Establishment of symbiosis:
• Establishment of rhizobium inside the host root and formation of root nodule is a
complex process and takes place in different events such as recognition and
infection of host root, differentiation of nodules, proliferation of bacteria into
nodules
1. Host specific and curling of root hairs: Rhizobium spp. recognize its host. Host
plants secrete exudates in the rhizosphere and compatible strains of Rhizobia
are stimulated over the other microbes in soil. Root exudates contains growth
stimulating substances like biotin, thiamine, amino acids etc. Bacteria grow
near the root surrounded by mucigel. Root curling occurs due to secretion of
Rhizobium which contains cytokinin and polymixin B
2. Infection of the root hairs: infection occurs due to invagination of the hair cell
walls in the region of curling
3. Nodule formation: as the infection continues through the root tissue, inner
cortical cells are stimulated by bacteria through hormone to divide and form an
organized mass of infected tissues which protrude from the root surface as
visible nodule. Inside the nodule, bacteria multiply and finally enlarged nodule
is formed containing Bacteroides
4. Nodule development and maintenance: the development and maintenance is
brought about by the proteins known as nodulins. Various factors such as
concentration of inorganic nutrients, soil temperature, light and shedding, CO2
concentration, rhizosphere microbes affect the formation and longevity of
nodules in roots of leguminous plants
Root nodule formation:
Bacterization:
• The technique of seed dressing with bacteria like Azobacter, Bacillus, Rhizobium
etc. it has been proved that bacteria can be established in the root region of the
plants to improve the growth of host plants
• Bacteria fertilizers such as cells of Azobacteria and phosphobacteria have been
used in many countries
• Bacteria used for bacterization:
Pseudomonas- rice
Rhizobium- leguminous plants
Azobacter- sugarcane
Bacillus- cereals, grass
• Bacteria which are used as biofertilizers are produced in large scale and should be
provided to the farmers
Method of mass cultivation:
Sterilized media is inoculated with the organism

incubated for 3-4 days at 30-32°C

Transfer to the larger fermenter and grow

Quality of broth will be checked

Broth is blended with sterile carrier(farmyard manure and charcoal powder)

Culture is packed in polyethene bags and kept at 25°C

Quality of carrier culture is checked

Stored in 4°C in controlled temperature and supplied to the farmers

Method of seed inoculation with Rhizobial culture:
• Seeds inoculated with rhizobial aqueous suspension during sowing has enhanced
the luxurious nodulation and good yield of crops as the rhizobium helps to fix the
atmospheric nitrogen which is helpful for the fertility of soil
• The steps for seed inoculation with rhizobial culture are:
Take about ½ lit water in a container

Add 10% sugar and boil it for 15 minutes then cool

10% gum Arabic solution can also be added which help as sticker for
rhizobium cells to seeds

Transfer the inoculum slurry on seeds and mix properly

Spread the seeds in shade for drying on cement floor or plastic sheets
• Some precautions to be taken while using rhizobial cultures:
- use of culture before expiry date
- use of small amounts of pesticides when required
- immediate sowing of seeds after mixing
- seeds must be stored at 40°C when not used immediately
Pelleting:
• The process in which the rhizobial cells are protected by using special methods of
inoculation when soil has adverse conditions such as dryness, acidity, excess
fertilizers and pesticides
• High amount of gum Arabic or carboxymethyl cellulose is added in the inoculum
slurry before mixing
• Pelleting agent is used when inoculated seeds are moist to get the seeds evenly
coated (pelleting agents such as: calcium carbonate, charcoal powder etc. are
used)
Green manuring:
• It is a farming practice where leguminous plants are grown in field then ploughed
into the soil and non-legume plants are cultivated and allowed to take benefits of
the already fixed nitrogen
• Various leguminous plants used as green manure are:
- Crotalari juneca, C striata, Cassia memosoides, Sebania aegytics
Advantages:
• Cheaper
• Pollution free and minimize pathogenic microbe in soil
• In addition to nitrogen, it also provide organic matter
Cyanobacteria inoculants:
• Cyanobacteria are blue green algae, photosynthetic, prokaryotic which fix N2 non-
symbiotically
• Examples are: Anabena, Nostoc and plectonema etc.
• They multiply in water logging condition, fix atmospheric N2 and release it into
the surroundings in the form of amino acids, proteins and other growth
promoting substances
• Cyanobacteria accumulate biomass which improves the physical properties of soil
and are useful in reclamation of alkaline soil
• Algalization:
• The process of application of blue green algal culture in field as biofertilizers
• Mass cultivation of cyanobacterial fertilizers:
• Four methods are applied for mass culture: cemented tank method, shallow
metal trough method, polythene lined Pit method and field method
• Mass cultivation of cyanobacteria can be done using any one method under the
following steps:
• Prepare the cemented tanks, shallow trays of iron sheets or polythene lined pits
in an open area(with should not be more than 1.5m)
• Transfer 2-3 kg of soil and add 100 gm of superphosphate. Water the pit about 10
cm height
• When water becomes clear, sprinkle 100 gm starter inoculum on the surface of
water
• When temperature is in between 35-40°C during summer, optimum growth can
be achieved
• After drying, the algal mat will get separate from the soil and forms flakes
• These are collected, powdered and kept in sealed polyethene bags and supplied
to the farmers
Azolla:
• It is aquatic fern which contains an endophytic cyanobacterium Anabena azollae
in its leaf cavity
• It can be widely used in rice fields
• Some examples of Azolla as biofertilizers: A caroliniana, A Mexicana, A nilotica
• Mass cultivation of Azolla:
Micro plant(20m2)
H2O(5-10cm)
P2O5(5-20 kg)
Azolla
incubation(3 weeks)
formation of Azolla mass

harvesting Azolla inoculum ready for use

Mycorrhiza as biofertilizer:
• Mycorrhiza is the symbiotic association of fungi with roots of plants. So, that the
nutrients absorbed from soil by the fungus are released to the host cells and in turn the
fungus takes its food requirements from the host.
• Mycorrhiza are of three types:
1. Ectomycorrhiza
2. Endomycorrhiza
3. Ectendomycorrhiza
• Ectomycorrhiza:
• The fungus which is located at the surface of the root is known as ectomycorrhizal
• Basidiomycota and include common woodland mushrooms, such as Amanita spp.
Boletus spp. and Tricholoma spp.
• It is found on the surface of the forest tress
• They absorb nitrogen, phosphorus, potassium and calcium
• They also convert complex organic molecules into simpler available forms, protect the
roots from the pathogens and produce growth promoting substances (Cytokinin)
Endomycorrhiza:
• The fungus which is located inside the roots is known as endomycorrhiza
• They are found in the roots of the most fruits and other horticultural
crops(coffee, pepper, cardamom etc.)
• They help in phosphorous nutrition
• They also produce growth promoting substances and offer resistance against
pathogens
• Fungus strains of Aspergillus, Azospirillum, Acomycota spp etc are used as
endomycorrhiza
• Ectendomycorrhiza:
• Shows the characteristics of both ectomycorrhizal and endomycorrhiza
• For examples: Phialophora finlandia, Chloridium paucisporum
• Hyphae are produced during this process
• Ectotrophic fungal infection generally occurs in higher plant species which are
very stout bushy type
• Mark classified(1991) mycorrhiza into seven types on the basis of relationship
with the hosts:
I. Vesicular arbuscular mycorrhiza(VAM)
II. Ectomycorrhiza
III. Ectendomycorrhiza
IV. Arbutoid mycorrhiza
V. Monotropoid mycorrhiza
VI. Ericoid mycorrhiza
VII. Orchidaceous mycorrhiza
• Among these VAM is commonly used as biofertilizers.
• VAM can be produced on a large scale by pot culture technique
• This requires the host plants, mycorrhizal fungi and natural soil
Production of VAM inoculum for application in fields:
Sterile pots soil

sterile sand: soil(1:1)

Sterile soil + VAM VAM spores in
spores watch glass
transfer
mix well
grow host seeds VAM spores
keep in glass houses

Young seedlings

after few weeks remove the seedlings gently

seedlings

check VAM spores microscopically and if present chop the roots

 Chopped roots as
starter inoculum

put small amount of starter inoculum 1 inch below

soil layer in the pots and sow host seeds in its vicinity
Inoculate in larger
pots

remove seedlings after 3-4 months

Inoculum in bulk
pack in polyethene bags and use in fields
Benefits from mycorrhiza to plants:
• They increase the longevity of roots and also the surface area of roots which help
to increase the rate of absorption of nutrients from soil and enhanced the plant
growth
• Some of the trees like pines cannot grow in new areas unless soil has mycorrhizal
inocula because of limited root hairs
• VAM enhances H2O uptake in plants
• VAM fungi reduces plant response to soil stress such as high salt levels, toxicity
associated with metals
• Some of them produce metabolites which help in vegetative propagation
• Increase the resistivity of plants to plant pathogens
Benefits from biofertilizers:
• It is cheaper and easy technique which can be used by small and marginal
farmers
• It is pollution free and increase soil fertility
• Azotobacter and Azospirillum supply nitrogen to soil as well as secretes
antibiotics which act as pesticides
• Using algal biofertilizers there is increase in rice yield
• Cyanobacteria secrete growth promoting substance amino acids, proteins,
vitamins etc which add sufficient amount of organic matter in soil
• Azolla supplies nitrogen, increase organic matter and fertility in soil and shows
tolerance against heavy metals
• Biofertilizers increase physiochemical properties of soil (soil texture, water
holding capacity, PH)
• Increase resistivity of plants to plant pathogens
Mushroom culture:
Mushroom culture:
• Mushrooms are edible food supplying a great array of nutrients
• They are the members of higher fungi of the class Ascomycetes and
Basidiomycetes
• They are heterotrophic in nature and have higher content of protein
• They have several beneficial role, so they are produced commercially
• Some wild mushrooms are poisonous such as Amanita verna, A virso
• Some edible mushrooms are: Agaricus bisporus, Heterbasidium annostum
• Advantages of mushroom:
• They utilize substances of poor value and are easy to produce
• They are delicious to eat and have good flavor
• They are rich in protein
• They are rich in vitamins particularly nicotinic acid and riboflavin and their
vitamins are well preserved during cooking, drying also
• They are devoid of starch which makes them suitable for diets of diabetic patients
• Various antioxidants have been identified in mushroom and they are used as
medicines in diseases like cancer
• The compost remained after mushroom cultivation can be used for biogas
production
• Cultivation of mushroom:
• Following steps are involved in mushroom cultivation:
1. Obtaining pure culture of mushroom
2. Preparation of spawns
3. Formulation and preparation of composts
4. Spawning spawn, running and cropping
Flow chart for cultivation of mushroom:
Formulation and Pure culture
preparation of compost

Stock culture

Compost spread over

beds
Spawn preparation
spawning

Spawn running

relative humidity, oxygen and marketing

temperature
Mushroom harvesting

canning
1. Obtaining pure culture of mushroom:
• To obtain pure culture mushroom are isolated from nature, purified and
characterized in laboratory
• They can be isolated in the PDA or Malt extract medium or can be obtained by
tissue culture technique
2. Preparation of spawns:
• Spawn is fungal growth medium impregnated with mycelial fragments of
mushroom which serves as inoculum for mushroom cultivation
• Many substrates are used for spawn making either alone or in combination, i.e.
rice straw cuttings, cotton wastes, grains of sorghum and rye
• The steps of grain spawn or straw spawn preparation are:
• Cooking the grains in water until they swell
• Cutting of straw into 5 cm long pieces and soaking into H2O for 5-10 minutes
• Decantation of water and mix 2% calcium carbonate
• Transfer into glass tube flasks
• Plugging with cotton
• Autoclave and cooling to 30-40°C
• Inoculating the substrate with pure culture of mushroom
• Incubation at suitable temperature
3. Formulation and preparation of compost:
• Methods of preparation of composts for mushroom cultivation are known as
composting
• The purpose of compost preparation is to provide medium for the rapid growth
of mycelium
• The physical and chemical compositions are developed in such a way that alter
the gross microbial community and promote maximum growth and yield of
mushroom
• Various substrate can be used (wheat straw, chicken manure, brewer’s grain,
urea, gypsum, horse manure)
• Substrates are filled in small trays or wooden boxes to make beds
• Trays or plastic bags are transferred in the room for its partial pasteurization at
low temperature
• Room temperature is maintained about 50-55°C for about 6 hrs.
• Later on, it is cooled down to a preferable temperature for casing the compost
• Casing is the covering of compost when spreads over the beds within layers of
soil or soil like materials
• It gives support to mushrooms, maintain temperature and prevents drying the
compost
4. Spawning, spawn running and cropping:
• Inoculation of compost in beds by spawn is known as spawning
• Bed material is inoculated by a small amount of spawn by removing it from
container and spreading over the bed material
• Room temperature and humidity is controlled for maximum mycelial growth and
spawn running
• After 10-15 days, compost is covered with mycelial growth
• Trays are watered continuously and after 1 month, mushroom crop become
mature at different intervals producing flushes
• Matured ones are picked and are marketed or canned
Composting:
• Composting is an aerobic process involving the activities of the microorganisms
under controlled conditions which result in the bioconversion of organic wastes
• Two types of microorganisms are involved: mesophilic and thermophilic
• The final product will be CO2, water, minerals and stabilized organic matter
• Various organic materials can be used for composting such as fruits, vegetables
scraps, egg shells, leaves, grass, kitchen wastes etc
• During composting, the complex substances are converted to simpler ones such
as nitrate, nitrite, CO2, methane, H2O, H2S
• The manure value depends upon the raw materials used and the extent of
decomposition by soil microorganisms
Application of composting:
• Treatment of organic wastes such as leaves, hazardous wastes, sludges
• Production of nutrient enriched compost to be used in agriculture, forestry
• Production of selective substrate for cultivation of mushroom

• Microorganisms used in composting are:

• Bacteria: Bacillus spp, Flavobacterium spp, Acromobacterium spp,
Thermoactinomycetes spp, Actinomyces spp, Pseudomonas spp
• Fungi: Alternaria spp, Aspergillus spp, Cephalosporium spp, Fusarium spp, Mucor
spp, Rhizopus spp
Advantages of composting:
• It is simple biological process
• Reduces the amount of solid wastes
• Can be used as bio-fertilizers
• It improves the plant growth
• It is more economic and pollution free process
• Composting process:
• The phases of composting are:
1. Material preparation
2. Decomposition
3. Finished product
• Based on characteristics of the composting, there are four different stages:
1. The initial stage, where mesophilic microbial activity bacteria is predominant
2. Mesophilic bacterial activity results in the rise of temperature. The rise in
temperature favors the activity of thermophiles
3. The cooling period where again mesophilic activities are regained
4. The maturity stage which leads towards the stabilization of the decomposed
product i.e. humus
Factors affecting composting:
I. Moisture
II. Aeration
III. C:N ratio
IV. Temperature
V. PH
I. Moisture: 60-70% of moisture is desirable for proper decomposition of organic
substrate
II. Aeration: it requires sufficient oxygen. O2 supply rate depends upon the
diffusion potential and process management. Diffusion rate depends upon the
porosity, particle size, moisture content
III. C:N ratio: The C:N ratio of substrate determines the effectiveness of
biodegradation. The C:N ratio should be 30:1 for suitable microbial growth
IV. Temperature: mesophilic and thermophilic microorganisms are responsible for
composting process
V. PH: it changes with the period of composting. Appropriate PH should be
maintained for the proper composting process
Methods of composting:
1. Static piles
a) pit method
b) Heap method
c) High temperature composting
2. Aerated piles
3. Continuous feed reactors
1. Static piles:
• It is simple and slow process. Under favorable condition, self heating in static
piles typically raise the temperature inside the compost piles to 55-60°C or above
in 3-4 days. The following are the types of piles:
a. Pit method:
• In this system, pits of size 1-2m wide and 1m length are dug and various
agricultural wastes are added layer by layer in the pit and decomposed for a
period of time
• The bottom layer consists of green plants and aquatic weeds available on the
farm followed by slit straw mixture and animal excreta. The layer is progressively
made until the pit is filled
• The top most layer is covered with mud and H2O and 4cm is maintained which
helps to reduce losses of Nitrogen that has been produced during composting
• In 10 weeks time, the pit materials are turned over or mixed with phosphate
compounds and H2O
• At the end of three months, the compost is ready for use on the farm
• For efficient composting, either compost from previous batch or microbes are
added in the compost piles
• The compost prepared is kept in moist condition
b. Heap method:
• In the heap system, decomposition is carried out 1 or 2 m above the ground
• The pile of dumping wastes occur in a cone or a pyramidal shape on growth
• These may or may not turn mechanically
• The heap is same as pile but larger mass is dumped
• In this technique, base material consists of cotton stalks followed by layering with
farm residues such as leaves, hay and garbage
• After wetting with H2O, the heaps are plastered with mud or plastic cover and
allowed to sit within a period of 2-3 months
• If necessary, the materials are turned and stirred again and covered with mud
and plastic cover
• The final product is decomposed organic matter
c. High temperature composting:
• This method utilizes soil, kitchen wastes, animal wastes, green leaves and many
other degradable wastes
• The base material consists of hard stalks of crops and this is followed by layering
with horse dung, soil, superphosphate and H2O is added at optimum level and
covered with mud
• Within 5 days, the temperature reaches 60-70°C
• Bamboo or maize stalks are inserted into mud-covered heap up to the bottom
and withdrawn after a day to leave behind the holes for ventilation
• Aeration is an important factor and it reduces the high temperature and keeps
the temperature balanced of the whole unit
• In every 2 weeks, the plaster is broken to mix up the piled up composting
material and the heap is again re-plastered
• After 2-3 months, composting process is completed
2. Aerated piles:
• The aerated piles process achieves faster composting rate through improved
aeration
• The design achieves partial oxygenation and involves suction of air through
perforated pipes buried inside compost

3. Continuous feed reactors:

• This is a rapid process and forms and uniform and stable product but requires
high investment
• It is used in a commercial scale only
• In this technique, composting in the reactor is accomplished in 2-4 days
• After processing in the reactor, the product requires curing time for about a
month prior to packaging and shipment
Tissue culture:
• Plant tissue culture is a technique of growing plant cells, tissues and organs in an
artificially prepared medium under aseptic conditions
• The term ‘’plant tissue culture’’ is used in broad sense that denote aseptic culture
of plant cells, tissues and organs
• The plant tissue culture is based on the principle of ‘Totipotency’
• CellularTotipotency means every living cell within the plant cells has potential to
generate into a whole plant, when provided suitable conditions
• Single cell without cell walls, pieces of leaves, stems, roots can often be used to
generate a new plant on culture media given the required nutrients and
hormones
• The plant part obtained from a plant to be cultured is called explant while the
main plant it obtained from is called mother plant
• Explant can be taken from different plant parts such as roots, shoots, leaves,
stems etc
• Advantages of tissue culture:
• Mass production of plant regardless of weather, time, space
• For the production of virus free plant
• For the biosynthesis of secondary metabolites
• Widely applied in agriculture, forestry and industry
• To conserve endangered plant spp
• To conserve cells rather than plant for specific characteristics such as herbicides
resistance
• To produce disease free plants
• Quick production of matured plants
• Production of multiples of plants in absence of seeds
Classes of plant tissue culture:
1. Callus culture: culture of callus (cell mass) on agar media produced from one
explant of seedling or other plant source
2. Cell culture: culture of cell in liquid media usually aerated by agitation
3. Organ culture: aseptic culture of embryos, anthers, ovaries or other organs on
nutrient media
4. Meristem culture: aseptic culture of shoot meristem or other explant tissue on
nutrient media to get complete plants
5. Protoplast culture: the aseptic isolation and culture of plant protoplast from
cultured cells or plant tissues
Requirements and steps involved in plant tissue culture:
• Requirements:
I. Media preparation area/room
II. Culture media washing powder, liquid detergents, disinfectants
III. Aseptic transfer chamber area
IV. Environmentally controlled culture room for growth and development of
cultures
V. Analytical rooms having equipments such as microscope, colorimeter, low
speed centrifuge, gas outlets etc
VI. Acclimatization room for the adjustment of plantlets
• Steps:
I. Preparation of culture media
II. Preparation of explants and sterilization
III. Inoculation of explants into the media
IV. Incubation of the inoculants
V. Acclimatization
VI. Field trial
1. Preparation of culture media:
• The media used provides nutritional requirements required for the growth of the
cell in vitro. The nutrient media is consist of variety of substances such as
inorganic salts, sucrose, vitamins, growth hormones and a few amino acids
• Inorganic minerals: it includes macro elements such as N, P, Ca, K, Mg and S in
the form of salt in large amounts(>0.5mm/lit) and the microelements such as Mo,
Cu, Zn, Mn, Fe and Cl (<0.5mm/lit)
• Organic materials: organic compounds serve as source of carbon and energy.
They are used in high concentration (20-50mg/lit). Sucrose and D-glucose are
commonly used as carbon source
• Complex organic additives: in some cases, complex additives such as peptone,
yeast extract, coconut milk, malt extract, tomato juice etc are used to support
plant tissue growth. Such additives should be used only when synthetic media fail
• Growth hormones: several growth hormones are known which stimulate the
biological activity in cultured materials. Auxin - to support cell division and
rooting etc in the form of IAA(indole 3 acetic acid), IBA( indole 3 butyric acid),
NAA(Napthalene acetic acid). Cytokinins- promotes cell division, regeneration of
shoot in the form of kinetin, BAP( benzyl amino purine)
• Others:
• Abscisic acid for shoot and bud regeneration
• Gibberlins: promotes shoot elongation
• The proper use of these hormones depend on the type of growth such as callus
formation, shoot formation, root formation
• Vitamins:
• These are required in trace amounts as they catalyze the enzyme system of the
cells
• Vitamin B1(most common) and other groups are: Vitamin B12, B6,Vitamin A,
Vitamin H etc are used
• Amino acids: various amino acids and amides are used. Widely used amino acids
are: L-aspartic acid, L-glutamic acid, L-asparagin, L-arginine etc
• Agar: used to solidify media(0.6-0.8%)
• PH: maintained at 5.7
• The various types of media available in the market are: B5 medium,
ER(Erikson)medium, Murashige skoog medium etc
2. Preparation of and sterilization of explant:
• The small piece of viable tissue isolated from the parent plant is called explant
which have the capability of cell growth, cell division and cell elongation
• Explant can be prepared from the stems, tuber, roots, buds, leaves etc
• The size of the explant may be 2-3 mm
• Surfaces of plant parts carry wide range of microbial contaminants, fungi and
bacteria being the common
• To avoid microbial growth which is detrimental to culture growth, explant must
be surface sterilized in disinfectant solution before planting in the nutrient
medium
• Commonly used disinfectants are: Calcium hypochlorite, sodium hypochlorite,
hydrogen peroxide, ethyl alcohol, silver nitrate, mercuric chloride, antibiotics
• After surface sterilization, plant material is washed with sterile distilled water for
3-4 minutes. All the processes are carried out under aseptic conditions under the
hood of laminar flow cabinet
3. Inoculation of the explant into the medium:
• During inoculation of explant into the solid medium, the medium kept in petri-
plates or test tubes or flasks should be held horizontally to reduce contamination
• The explant is pushed half way into the agar
• The growth of the explants depends upon the media having different
concentrations of growth hormones as Auxin, cytokinin or both which ultimately
give rise to shoot, root and callus
4. Incubation of the inoculants:
• The media after inoculation is kept into the incubator or room which is already
maintained at 24-26°C and the proper light should be provided for 4-5 weeks
• After getting the suitable conditions, the plantlets develop that should be
subjected to acclimatization
5. Acclimatization:
• It is the process of adjustment of the plantlets developed in the laboratory to the
outside of the lab
• Therefore, they can adjust later in the field conditions, for this the plantlets are
taken out from the rooting medium which is washed slowly with the running
water for an hr. to ensure that no piece of agar is left on the surface of the
plantlets
• If agar remains there, it would increase the chance of attacks by microbes
• Now the plantlet is put into a low minimal salt medium for 24-48 hrs. and then
transfer to a pot that contain autoclaved sterilized mixture of clay soil
• The plant in this condition should be left for 15-20 days
• At this condition, the plant is fully acclimatized and is able to withstand in open
environmental conditions
5. Field trial
• After acclimatization, plant is transferred to soil as in normal agriculture
techniques
• Types of tissue culture:
1. Cell culture:
a) Isolation of single cell:
• single cell can be isolated either from cultured tissue or from intact plant organs.
Isolation can be achieved by either mechanical method or enzymatic method
• For mechanical isolation, cultured tissues or plant organs are cut into small pieces
(1cm2). ground into a suitable culture medium using pestle and mortar, the
resulting homogenous is filtered through muslin cloth and the cells are pelleted
out by centrifugation
• In the enzymatic method, the lower epidermis of the leaf is peeled off and the
leaves are cut into moderate pieces(4cm2) which are incubated in pectinase
solution
• Single cells can also be isolated from suspension culture by filtering out cells
clumps and harvesting the cells by centrifugation
b) Culture of single cells:
• Various methods have been developed to grow individual cells on a nutrient
medium. The methods are: Bergmann’s cell plating technique, micro chamber
technique, micro drop technique etc
• Bergmann’s cell plating technique is most widely used and in this method, free
cells are suspended in a large medium, later mixed with an equal volume of
molten, cooled, agar medium and platted in a petri plate
• The petri plates are sealed with paraffin and incubated in a dark at 25°C for 3-4
weeks
• Colonies or calluses will be observed on the agar surface after 3-4 weeks after
incubation
c) Suspension culture:
• Cell suspension is prepared by transferring a fragment of callus to the liquid
medium which is continuously agitated to obtain a suspension culture
• Agitation of pieces break them into smaller clumps and single cells and also
maintains uniform distribution of cells and cells clumps in the medium
• It also allows gaseous exchange
• When cells are transferred to the suitable medium, they divide after lag phase
and linearly increase their population
• After sometime, due to depletion of nutrients, the rate of cell division decreases
until it comes to the stationary phase
• At this stage it is essential to subculture the cell to keep them viable
• Properties of cultured plant cell suspension common with culture of
microorganisms:
 They grow in a sterile environment
 They are homologous in size
 They have doubling time which is longer than that of microorganisms
 they can grow in a large scale
Applications of cell culture:
a) Mutant selection: by using cell culture, mutant cells resistant to antibiotics,
herbicides, fungal toxins can be selected
b) Production of secondary metabolites: plants are important sources of varieties
of chemicals used for varieties of purposes such as pharmacy, medicine and
industry. In recent years, plant cell suspension are used for the production of
these chemicals on a commercial scale. The secondary metabolites include
flavors, perfumes, agrochemical etc.
c) Biotransformation:
• In this technique low cost precursors are used as substrate and are transformed
into value added high cost products
• For example, Datura cell culture posses ability to convert Hydroquinone into
arbutin(used as urinary antiseptic)
d). Single cell protein:
• Single cell cultures are also being used for production of single cell protein
2. Organ culture:
• Root, shoot, leaves are the organs can be used in plant tissue culture
a). Root culture: generally excised is cultured in liquid medium. Root of several
species of gymnosperm and angiosperm have been successfully cultured
b). Shoot culture:
• in shoot culture apical meristem is cultured. Because of culture of large shoot
this technique is also called meristem culture, meristemming and mericlones.
• Shoot tip culture is extensively used in agriculture, horticulture and forestry.
• The shoot tip may be cut into fine pieces to obtain more than one.
• Plantlet from each shoot tip. Generally explants taken from actively growing
plants at the beginning of growing seasons are the most suitable
I. Culture medium: in general MS medium has been found satisfactory for the
most plant species. MS medium of low concentration is suitable. Growth
regulator requirements depends upon the stages of culture processes
II. Environment during culture: during culture initiation and shoot multiplication
phases, 25°C temperature and light intensity of 1000 lux and during root 3000-
10,000 lux
iii. Rooting of shoot: rooting medium has low salt and reduced sugar levels.
Rooting takes about 10-15 days. Plantlets with 0.5- 1.0 cm roots are transplanted
into pots
Iv. Transfer of plantlets to soil
3. Pollen culture:
• Isolated pollen grains when cultured in vitro, give rise to haploid embryos, this
approach is called pollen culture
• The aim of pollen culture is to get haploid plant by induction of embryogenesis
• Haploid chromosome has single complete set of chromosome that in turn may be
useful for the improvement of many crop plants
• Successful pollen culture is reported for several species such as potato, rice,
maize etc.
Steps:
• Dissected anthers are taken in a sterile beaker containing liquid media and
passed with a glass rod to allow pollen squeeze out
• The suspension with anthers and pollens is filtered through a nylon mesh of
suitable size that allows only pollen to pass through
• The filter is centrifuged at 500-800 rpm
• The pollen pellet is collected and suspended into liquid medium which are
inoculated on a solid or in a liquid medium maintained at 25°C. the pollen may
develop directly into embryo within 15 days and follow one of the several indirect
pathway to produce haploid plantlets
4. Protoplast culture:
• Protoplast is biologically active and most significant materials of cells
• Protoplasts are isolated from cells by two methods:
1. Mechanical method: the cells are plasmolysed in mannitol solution for 3 hrs. causing
the protoplast to shrink away from the cell wall, then the protoplasts are removed
from the cell through incision
2. Enzymatic method: protoplasts are usually isolated by treating tissues with a mixture
of cell wall degrading enzymes (cellulase, hemicellulase, pectinase, protease etc.) in
solution which contain osmotic stabilizers to preserve the structure and viability of
the protoplast. Then the solution is filtered and centrifuged
• From the protoplast, solution of known density about 1ml suspension is poured on
sterile and cooled nutrient medium in petri dishes. The plate are incubated at 25°C in a
dim white light
• After 7-10 days of protoplast culture, the protoplast regenerates a cell wall, under goes
cell division from callus. The callus also can be sub-cultured and embryogenesis begins
from callus, when it is placed in nutrient medium. The embryo develops into seedling
and finally matured plants
Importance of protoplast isolation and culture
• To develop new hybrid plant of desired characteristics through protoplast fusion
and genetic engineering
• This helps in crop improvement by somatic hybridization and cell modification
• The protoplast in culture can be generated into an entire plant
• The technique in future will be one of the most frequently used research tools for
tissue culture
• Protoplast fusion(somatic cell hybridization):
• Production of hybrid plants through the fusion of protoplast of two different
species or varieties is called somatic cell hybridization and such hybrids are
known as somatic hybrids
• Fusion of cytoplasm of two protoplasts result in cohesion of cytoplasm
• The nuclei of two protoplasts may or may not fuse together even after fusion of
cytoplasm
• The binucleate cells are known as heterokaryon or heterocytes
• When the nuclei are fused the cells are known as hybrids or synkaryocytes and
when only cytoplasm fuse and genetic information from one of the two nuclei is
lost, the cells are called cybrid or cytoplasmic hybrid or heteroplast
• There are some genetic factors which are carried in cytoplasmic genes (such as
susceptibility, resistance to some of the pathotoxins and drugs)
• Therefore, production of cybrid which contain the mixture of cytoplasm but only
one nuclear genome can help in transfer of cytoplasmic genetic information from
one plant to another
Steps for somatic hybridization:
1. Isolation of protoplast from suitable plants
2. Mixing of protoplast in centrifuged tube containing chemicals which promote
protoplast fusion(polyethylene glycol, sodium nitrate maintain high PH(10.5)
and temperature of 37°C and as a result of fusion of protoplast viable
heterokaryotes are produced
3. Wall regeneration by heterokaryotic cells
4. Fusion of nuclei of heterokaryon to produce hybrid cells
5. Planting and production of colonies of hybrid cells
6. Regeneration of plants
7. Selection and characteristics of hybrid or cybrid plants
• Somatic hybridization has been considered special significance in improvement of
crops such as banana, potato, sugarcane etc. in which sexual reproduction is
weak or absent
• Pomato, the somatic hybrid between potato and tomato
Fusion of protoplast of potato, tomato and production of hybrid
plant
Potato plant Tomato plant

Protoplast Protoplast

fugigenic chemicals for protoplast fusion

Heterokaryon
planting of fused protoplast
Growth of hybrid colonies
selection of hybrid colonies and planting on medium
Hybrid callus
transfer callus to nutrient medium
Hybrid plants(pomato/topato)
Micro-propagation and disease free plants:
• Micro-propagation means production of large number of plantlets when the
shoot tip is used as an initial material by the technique of tissue culture
• The principle of micro-propagation is based on the phenomenon that shoot tip
when cultured on tissue culture medium can develop large number of shoot
identical to parent plant
• All the plants produced possesses similar phenotypic and genotypic character
• In crops, especially those propagated by vegetative means, the systemic
pathogens are transmitted through the propagules (root, tuber, bulb etc.)
• It has been observed that plants raised in culture using apical meristem of
infected plants are free of pathogens
• Using this technique several important plants have been mass propagated
Steps in micro-propagation:
• Generally, the sterilized shoot tips or axillary buds are taken for micro
propagation
• Following steps are involved:
• Stage I – establishment of tissue in vitro
• Stage II – multiplication of shoots
• Stage III – root formation and conditioning of propagules prior to transfer to
green house
• Stage IV – growth in pots followed by field trials
• Advantages of micro-propagation:
• Rapid multiplication of superior clones and maintenance of uniformity
• Multiplication of disease free plants
• It is an alternative technique of vegetative propagation
• Minimum growing space is required in nurseries
Selection of meristem for micro propagation:
• Meristem parts are always in the state of continuous cell division which is rapid
than multiplication of virus to prevent from virus to reach that can cause
infection
• There is no development of vascular tissues in apex i.e. absence of transporting
tissues due to which circulation of viruses doesn’t occur
• It may be exposed to 30-40°C to inactivate the virus
• General concept of cell fusion and embryo transfer:
• Cell fusion means the fusion of two somatic cells
• The fused cells can independently regenerate and develop into complete plant if
provided with suitable growth conditions
• Many plants seeds are difficult to germinate, for such, embryo transfer technique
is applied, by using embryo transfer technique, clones of particular plants can be
developed
• These are genetically identical to parent plant and among themselves also
• By embryo transfer, many endangered species of plants can be preserved by
establishment of embryo bank
• Application of tissue culture:
• Improvement of hybrid through cell fusion and hybridization technique
• Production of encapsulated(artificial) seeds
• Production of virus free toxic resistant plants
• Micro-propagation of large scale production of plantlets
• Production of secondary metabolites
• Transgenic plants for crop improvement
• Molecular farming from transgenic plants such as production of edible vaccines,
edible antibodies
• Shortening of breeding cycle
Single cell protein:
Single cell protein:
• cell proteins are the dried cells of microorganism, which are used as protein
supplement in human foods or animal feeds.
• Microorganisms like algae, fungi, yeast and bacteria, utilize inexpensive feedstock
and wastes as sources of carbon and energy for growth to produce biomass,
protein concentrate or amino acids
• Since protein accounts for the quantitatively important part of the microbial cells,
these microorganisms, also called single cell protein as natural protein
concentrate.
• With increase in population and worldwide protein shortage the use of microbial
biomass food and feed is more highlighted.
Raw materials for SCP production:
• Production of SCP requires microorganisms that serve as the protein source and
the substrate i.e. biomass on which they grow
• The biomass can be plant biomass or organic biomass
• The microorganisms used belong to the group of algae, fungi and bacterium

• List of microorganisms used for SCP production:

• Fungi: Aspergillus fumigatus, A niger, Rhizopus cyclopium
• Yeast: Sccharomyces cerevisae, Candida tropicalis, Candida utilis
• Bacteria: Pseudomonas fluorescence, Lactobacillus, Bacillus megaterium
Biomass:
• It plays important role in the production of SCP
• Selection of biomass depends upon the microorganisms used for the production
• As for example, Algae are cultivated on sewage whereas yeast are cultivated on
agro-industrial wastes

Algal biomass:
• Algae grow auto-tropically
• Requires low intensity of light
• Temperature 35-40°C and PH 8.5-10.5
• Cultivated in large trenches of sewage oxidation ponds
Bacterial and fungal biomass:
• Bacteria and fungi can be grown in wide range of substrates
• They require minimum temperature of 15-34°C and PH 5.7
• Yeast biomass:
• Cultivated on agro-industrial wastes such as molasses, starchy materials, fruit
pulp etc.
• It requires a temperature of 30-34°C and PH 3.5-4.5
• It also requires addition of inorganic acids and Sulphur supplements in the form
of salts
• Factors affecting biomass production:
• Illumination time
• Temperature
• PH
• Suitable strains, agitation, sterile conditions
SCP production:
1. Selection of suitable strain
2. Fermentation
3. Harvesting
4. Post harvest treatment
5. SCP processing for food
1. Selection of strain:
• It is a very critical step as the quality of protein depends totally on the microbe
that is used for the production
• Therefore, careful selection of the strain should be done
• Care should be taken that the selected strain should not produce any toxic or
undesirable effects in the consumers
2. Fermentation:
• It can be carried out in the fermenter which is equipped with aerator, thermostat,
PH etc. or in the trenches or ponds
• Microbes are cultured in fed- batch culture
3. Harvesting:
• When the colonies of microbes are fully developed, they are harvested
• The bulk of cells are removed from the fermenter by decantation
4. Post harvest treatment:
• After harvesting, the cells are subjected to a variety of processes
• Post harvesting treatment includes steps like separation by centrifugation,
washing, drying etc.
5. Processing for food:
• It includes:
1. Liberation of cell protein by destruction of indigestible cell wall
A) Mechanical method:
• Crushing
• Crumbling
• Grinding
• Pressure homogenization etc.

B) Chemical method:
• Enzyme and salts are used to digest or disrupt the cell wall
• Salts like Nacl, sodium dodecyl sulfate etc where as nuclease enzymes are used
C) Physical method:
• Freeze- thaw
• Osmotic shock
• Heating and drying
2. Reduction of nucleic acid content:
• Chemical and enzymatic treatments are preferred
• Chemicals used are- acidified alcohol, salts and alkalis
• Use of such chemicals lead to formation of lygino-alanine which causes
hypersensitivity reactions
• Enzymes which are used include ribonuclease and nuclease enzymes
• These enzymes can be used exogenously or can be induced endogenously
Advantages:
• Rapid succession of generation
• Easily modifiable genetically
• High protein content of 43-85% in dry mass
• Broad spectrum of original raw material used for production, which also includes
waste products
• Production in continuous culture
• Consistent quality not depend on climate in determinable amount
• Low land requirement, economically beneficial
• Utilization of solar energy
• Cellular, molecular and genetic alterations
Applications:
• Used as protein supplemented food
• Also is the source of vitamins, amino acids, crude fibers
• Supplemented food for the undernourished children
• Controls obesity
• Provides instant energy
• Reduce body weight, cholesterol, and reduce blood sugar level in diabetic patient
• Increase lactation
• Useful for healthy eyes and skin due to presence of β-carotene
• Used for preparation of many herbal face cream
• Used for many herbal beauty products
• Excellent source of protein and used to feed cattle, fishes etc.
Biocontrol agents:
• Control of organism by organism
• Biological control is the reduction of the amount or disease producing activity of
a pathogen accomplished by one or more organisms other than man
• mechanism of biological control:
• Antagonism:
1. Competition: use and disuse of resources (space and nutrition) by one
individual(microorganism) that reduces the availability of that resources to
others
2. Antibiosis: condition in which one or more metabolites(antibiotic substances)
secreted by an organism which have harmful effect on the others. For example,
Trichoderma viride produces viridin
3. predation: relationship between organisms in which one organism capture and
feed on the other(predator prey relationship). For example, Dactylaria sp.
(predacious fungi) and Dactylella sp. (parasitic nematode), Lady bugs(larvae)
are predators of Aphids and mites
4. Parasitism: relationship between host and pathogen. For example, most
parasitoids are wasps. As for example, Ichneumonid wasp attack caterpillar
Methods of application of biological control agents:
1. Seed treatment
2. Soil treatment
3. Foliar spray
4. Root dip
Attributes of successful biocontrol agents:
• Must not be pathogenic to plants and animals
• Level of pathogen control must be high
• Should live longer in soil or host tissues
• Should have rapid reproducible capacity
• Should be a good competitor
• Should be capable of controlling more than one pathogen
• Should be suitable for long term storage
Bioinsecticides:
• Microorganisms used for insect control are often called bioinsecticides
• The term biopesticides is used for all biocontrol agents
• Viruses, bacteria, fungi, protozoa, mites are employed to control a variety of
insects attacking both plants and animals
• Microbial insecticides can be: microbially produced toxic substances or organisms
• Advantages:
• Organisms used in microbial insecticides are essentially non-toxic and non-
pathogenic to wild life, humans and other organisms
• The toxic action of microbial insecticides is often specific to a single group or
species of insects
• Most microbial insecticides can be used in conjunction with synthetic chemicals
insecticides
• Residues present no hazards to humans or other animals
• The pathogenic microorganisms can establish in a pest population or its habitat
and provide control during subsequent pest generation
• Microbial pesticides:
• Are the products derived from various microscopic organisms
• Microbial products may consist of the organisms themselves or metabolites they
produce
• It is divided into 6 subcategories of products:
- bacteria
- fungi
- protozoa
- viruses
- yeast
Bacteria:
• Are present in soil and are the most abundant in soil samples
• The most well known and widely used of all biopesticides are insecticides based
on Bacillus thuriengiensis(Bt)
• Bt provides insecticidal proteins (known as δ- endotoxin) that kills pest
• Fungi:
• Some spp of fungi are useful in microbial biopesticides
• They attack and parasitize plant pathogens
• Two most common fungal biopesticides are: Trichoderma spp and Beauveria
bassiana
• Trichoderma readily colonize plant roots, without harming the plants and helps
by antibiosis, competition, induce host resistance etc
• Beauveria bassiana is a fungus that acts as a parasite on many insect spp
Protozoa:
• They are single celled eukaryotic organisms present in water and soil
• The protozoan Nosema locustae is known to be a natural biocontrol agent of
many grasshoppers
• Nosema infects atleast 90 spp of grasshoppers
• It is non-toxic to humans and other mammals
• It infects and weakens young grasshoppers and adversely affects female
grasshoppers’ ability to reproduce
• Viruses:
• Baculoviruses are microbial pesticides and are family of naturally occurring
viruses known to infect only insects
• The specific in their action and kill only one or few spp of caterpillars
• The viral DNA replicates in the nuclei of host cells and then spreads throughout
the body of the larvae, turning it into a virus factory
• The infected insects stop feeding, within a few days dies and disintegrate
Yeast:
• A variety of yeasts are known to be useful in controlling plant diseases
• Non- pathogenic Cryptococcus and Candida spp naturally occur on plant tissues
and in water
• The yeasts serve as antagonist to fungal pathogens such as gray mold and blue
mold which cause post harvest decay
• There is evidence that it provides enzymes that can degrade fungal cell walls and
stimulate plant host defense pathways in freshly harvested fruit
Bioherbicides:
• It is a biologically based control agent for weeds and eco-friendly
• Agents used as bio-herbicides are:
• Insects
• Fungi
• Bacteria

• insects
• Caterpillars of the moth Cactobastis cactorum bore into the pads of prickly pear
• This damage the cactus and introduces a bacterium that causes the plant to die
• The alligatorweed fleabettle controls the floating aquatic weed
Fungi:
• Many fungi have been shown to exhibit broad spectrum weed control ranges
• Most often the organism used is a fungus, hence the term myco-herbicides is
used
Weeds pathogens(fungal herbicides)
Velvet leaf Collectotrichum coccodes, Fusarium lateritium
Wild coat Septoria tritici
Water hyacinth Alternaria eichhorniae
Bacterial herbicidal agents:
• Bacteria are also found to be useful in controlling weeds, as for examples:
Bacteria weeds
Xnathomonas campestris annual blue grass
Rhizobacteria downy brome
Advantages and limitations of biocontrol agents:
• Decreases disease intensity
• Reduces the use of chemical fungicides
• Reduce the undesirable effects from chemical pesticides
• Play a key role in integrated management of diseases
• Safe for the users and the farming community
• Provide natural long term immunity to crops and soil

• Deleterious effects on non-target microorganisms

• Pathogen may develop resistance to biocontrol agents
• Seasonal or weather phenomenon can make biocontrol agents ineffective
THANK YOU