Sinhgad College of Engineering, Pune -41

Department of Biotechnology

Third Year B. Tech Biotechnology

Agricultural Biotechnology 315465 (C)

List of Experiments:
1.​ Plant Genomic DNA Extraction using CTAB Method
2.​ Restriction Enzyme digestion of Plant DNA
3.​ Plant Tissue Culture Lab: Media Stock Preparation & Sterilization
4.​ Callus Induction from explant
5.​ Isolation of agriculturally important microorganisms –Trichoderma Spp.
6.​ Isolation of N2 fixers- Rhizobium spp, Azotobacter Spp.
7.​ Biofertlizer Production & Quality Test
8.​ Report for case studies

Dr Manisha Shinde
Subject Teacher
Sinhgad College of Engineering, Pune -41
Department of Biotechnology

Third Year B. Tech Biotechnology

Agricultural Biotechnology 315465 (C)

INDEX

[Link]. Experiment Date Remark

1 Plant Genomic DNA Extraction using
CTAB Method
2 Restriction Enzyme digestion of Plant
DNA
3 Plant Tissue Culture Lab: Media Stock
Preparation & Sterilization
4 Callus Induction from explant
5 Isolation of agriculturally important
microorganisms –Trichoderma Spp.
6 Isolation of N2 fixers- Rhizobium spp
7 Isolation Of N2 Fixing Bacteria -
Azotobacter From Soil Samples
8 Biofertlizer Production & Quality Test
9 Case Study Report
Experiment No.: ​ ​ ​ ​ ​ ​ ​ ​ Date:
Plant Genomic DNA Extraction using CTAB
Introduction
DNA extraction from plant tissue can vary depending on the material used. Essentially any
mechanical means of breaking down the cell wall and membranes to allow access to nuclear
material, without its degradation is required. For this, usually an initial grinding stage with liquid
nitrogen is employed to break down cell wall material and allow access to DNA while harmful
cellular enzymes and chemicals remain inactivated.
Once the tissue has been sufficiently ground, it can then be resuspended in a suitable buffer, such
as CTAB. One of the most commonly used methods to extract DNA from plants uses the ionic
detergent cetyltrimethylammonium bromide (CTAB) to disrupt membranes and a
chloroform-isoamyl alcohol mixture that separates contaminants into the organic phase and nucleic
acid into the aqueous phase. In order to purify DNA, insoluble particulates are removed through
centrifugation while soluble proteins and other material are separated through mixing with
chloroform and centrifugation. DNA must then be precipitated from the aqueous phase and washed
thoroughly to remove contaminating salts. The purified DNA is then resuspended and stored in TE
buffer or sterile distilled water. This method has been shown to give intact genomic DNA from
plant tissue.
To check the quality of the extracted DNA, a sample is run on an agarose gel, stained with
ethidium bromide, and visualised under UV light.

Materials:

Plant Leaf sample

CTAB buffer
Microfuge tubes
Mortar and Pestle
Absolute Ethanol (ice cold) 70 %
Ethanol (ice cold)
7.5 M Ammonium Acetate
55º C water bath
Chloroform : Iso Amyl Alcohol (24:1)
Water (sterile)
Agarose 6x Loading Buffer
1x TBE solution Agarose gel electrophoresis system
Ethidium Bromide solution
CTAB buffer (100ml )
2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide)
10.0 ml 1 M Tris pH 8.0
4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic acid Di-sodium salt)
28.0 ml 5 M NaCl
40.0 ml H2O
1 g PVP 40 (polyvinyl pyrrolidone (vinylpyrrolidine homopolymer) Mw 40,000)
Adjust all to pH 5.0 with HCL and make up to 100 ml with H2O.

1 M Tris pH 8.0
Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to 8.0 by adding 42 ml of concentrated
HCL. Allow the solution to cool to room temperature before making the final adjustments to the
pH. Adjust the volume to 1 L with H2O. Sterilize using an autoclave.

5x TBE buffer
54 g Tris base 27.5 g boric acid 20 ml of 0.5M EDTA (pH 8.0) Make up to 1L with water.
To make a 0.5x working solution, do a 1:10 dilution of the concentrated stock.

1% Agarose gel
1 g Agarose dissolved in 100 ml TBE

Procedure –
1.​ Grind 200 mg of plant tissue to a fine paste in 1 ml CTAB buffer.
2.​ Transfer 500 μl CTAB/plant extract mixture to a microfuge tube.
3.​ Incubate the CTAB/plant extract mixture for about 15 min at 55o C in a recirculating water
bath.
4.​ After incubation, spin the CTAB/plant extract mixture at 12000 g for 5 min to spin down
cell debris.
5.​ Transfer the supernatant to clean microfuge tubes.
6.​ To each tube add 250 μl of Chloroform: Iso Amyl Alcohol (24:1) and mix the solution by
inversion.
7.​ After mixing, spin the tubes at 13000 rpm for 1 min.
8.​ Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge tube.
9.​ To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice cold
absolute ethanol.
10.​ Invert the tubes slowly several times to precipitate the DNA.
11.​ Generally the DNA can be seen to precipitate out of solution.
12.​ Alternatively the tubes can be placed for 1 hr at -20 o C after the addition of ethanol to
precipitate the DNA.
13.​ Following precipitation, the DNA can be pipetted off by slowly rotating/spinning a tip in
the cold solution.
14.​ The precipitated DNA sticks to the pipette and is visible as a clear thick precipitate.
15.​ To wash the DNA, transfer the precipitate into a microfuge tube containing 500 μl of ice
cold 70 % ethanol and slowly invert the tube.
16.​ Repeat. ((alternatively the precipitate can be isolated by spinning the tube at 13000 rpm for
a minute to form a pellet. Remove the supernatant and wash the DNA pellet by adding two
changes of ice cold 70 % ethanol )).
17.​ After the wash, spin the DNA into a pellet by centrifuging at 13000 rpm for 1 min.
18.​ Remove all the supernatant and allow the DNA pellet to dry (approximately 15 min).
19.​ Do not allow the DNA to over dry or it will be hard to re-dissolve.
20.​ Resuspend the DNA in sterile DNase free water (approximately 50-400 μl H2O; the amount
of water needed to dissolve the DNA can vary, depending on how much is isolated).
21.​ RNaseA (10 μg/ml) can be added to the water prior to dissolving the DNA to remove any
RNA in the preparation (10 μl RNaseA in 10ml H2O).
22.​ After resuspension, the DNA is incubated at 65o C for 20 min to destroy any DNases that
may be present and store at 4o C.
23.​ Agarose gel electrophoresis of the DNA will show the integrity of the DNA, while
spectrophotometry will give an indication of the concentration and cleanliness.

DNA quality confirmation –

1.​ Prepare a 1 % solution of agarose by melting 1 g of agarose in 100 mL of 0.5x TBE buffer
in a microwave for approximately 2 min.
2.​ Allow to cool for a couple of minutes then add 2.5 μl of ethidium bromide, stir to mix.
3.​ Cast a gel using a supplied tray and comb. Allow the gel to set for a minimum of 20 min at
room temperature on a flat surface.
4.​ Load the following into separate wells o 10 μL 1kb ladder o 5 μL sample + 5 μL water + 2
μL 6x Loading Buffer
5.​ Run the gel for 30 min at 100 V – Expose the gel to UV light and photograph
(demonstration) Confirm DNA quality, presence of a highly resolved high molecular
weight band indicates good quality DNA, presence of a smeared band indicates DNA
degredation.

Result:

Signature:
Experiment No.:​ ​ ​ ​ ​ ​ ​ ​ Date:

Restriction Enzyme digestion of Plant DNA

The objective of this experiment is to demonstrate the activity of Restriction endonucleases

EcoRI and to analyse the results of a Restriction endonuclease digestion using ‘Agarose gel
electrophoresis’.
Type II endonucleases are commonly described as “restriction enzymes” which recognize
specific restriction DNA at a specific restriction site and cleaves double stranded DNA by
hydrolyzing tow phospho diester bonds within defined nucleotide sequences to generate
reproducible nucleotide fragments. Thus they are also referred as “molecular scissors”. The
DNA is the most widely used substrate for screening Restriction enzymes.
EcoRI is a restriction enzyme, which is isolated from [Link] RX13 bacterial cells. EcoRI
acts as a dimmer and recognizes six base pair palindromic sequences (nucleotide pair
sequences which are same when read forward or backward from a central axis of symmetry). It
acts on the phosphodiester bonds between two nucleotide and cuts at 5 recognition sites. This
staggered cleavage of double stranded DNA results is sticky/cohesive ends, which are
identical, complementary, single stranded projections and can base pair with each other.
In the experiment λ DNA is subjected to digestion by [Link] enzyme. The recognition
sequences for [Link] in λ DNA are ‘GAATTC’. EcoRI enzyme protein binds itself to the DNA
molecule and then recognizes specific restriction on the λ chromosome. E coRI cleaves ds
DNA strand between ‘G’ and ‘A’ nucleotides by hydrolyzing 2 phospho diester bonds (1 per
strand) within defined nucleotide sequences, and forms fragments with 5 terminal phosphate
and 3 terminal hydroxyl residue. This results in living 5’ overhang of TTAA on the
complimentary strand.
5’ – GAATTC – 3’ 5’ – G AATTC – 3’
3’ – CTTAAG – 5’ 3’ – CTTAA G – 5’
Before Digestion After Digestion

Classification of restriction endonucleases:

System Key Features
Type I One enzyme with different subunits for recognition, cleavage and methylation.
Recognizes and methylates a single sequence but cleaves DNA upto 100bp away.
Type II Two different enzymes which both recognize same target sequence, which is
symmetrical. The two enzymes either cleave or modify the recognition sequence.
Type One enzyme with two different subunits, one for recognition and modification and one
III for cleavage. Recognizes and methylates same sequence but cleaves 24-26bp away.
Type Two different ezymes but recognition sequence is asymmetric. Cleavage occurs on one
IIs side of recognition sequence upto 20bp away.

Materials:
DNA
Enzymes- EcoR I
10X assay buffer, distilled water
Procedure:
1. Make the additions as per the following table:
RESTRICTION ENZYME SET-UP:

Component
s Volume(μl)

DNA 10.0
Water 2.5
10X Assay Buffer 1.5
Enzyme 2-EcoR I 1.0
Total 15.0

2. Following additions, the mixture is set for incubation at 37ºC for 2 hrs.
3. The restricted DNA and vector are run on 0.8% agarose gel for confirmation.
Interpretation:
a) Complete digestion is indicated by the formation of 6 bands.
b) A single DNA band seen if there is no digestion.
c) Few extra bands are in addition to the expected pattern it the digestion is incomplete.

Result:

Signature:
Experiment No:​ ​ ​ ​ ​ ​ ​ ​ ​ Date:
Basics of Plant Tissue Culture
Plant Tissue Culture Lab: Media Stock Preparation & Sterilization

Objectives
1. To know the basics of plant tissue culturing.
2. To know the production of callus from explant.
3. Measure the efficacy of root and shoot.

Theory

The theoretical basis for plant tissue culture was proposed by Gottlieb Haberlandt, German
Academy of science in 1902 on his experiments on the culture of single cell. The first true cultures
were obtained by Gautheret from cambial tissue of Acer pseudoplatanus.

The term plant tissue culture (Micro propagation) is generally used for the aseptic culture of cells,
tissues, organs and their components under defined chemical and physical conditions in vitro. The
basic concept of the plant body can be dissected into smaller part termed as “explants” and any
explants can be developed into a whole plant. It is a central innovative areas of applied plant
science, including agriculture and plant biotechnology. This technique is effective because almost
all the plants cell are totipotent; In each cell possesses the genetic information and cellular
machinery necessary to generate the whole organism. Since, this technique can be used to produce
a higher number of plants that are genetically similar to a parent plant as well as to another.

Two concepts, plasticity and totipotency, are the central processes to understand the regeneration
and plant cell culture. Plants, due to its longer life span and sessile nature, have developed a greater
ability to overcome the extreme conditions. Most of the processes included in plant development
and the growth, adapt to environmental conditions. When the plant cells and tissues are cultured in
vitro, most of them are generally exhibit a very high degree of plasticity, which allows one type of
organ or tissue to be initiated from another type. Like this way, the whole plant can be
subsequently regenerated. These maintenance of genetic potential is called totipotency.

The plant tissue culture medium is an artificial nutrient supplement of organic and inorganic
nutrients used for cultivation of plant tissue media. The appropriate composition of the medium
largely determines the success of the culture. The culture media used for the in vitro cultivation of
the plant cells are composed of three basic components.

1) Essential elements (normal ions) supplied as a complex mixture of salts.

2) An organic supplements providing vitamins and amino acids.
3) A source of fixed carbon which is usually supplied as sucrose.

When cultured in an appropriate medium having auxin and cytokinin, explants will give rise to an
unorganized, growing and dividing mass of cells called callus. Callus cultures are initiated from a
small part of an organ or tissue segment called the explants on a growth supporting solidified
nutrient medium under sterile conditions. Any part of the plant organ or tissues may be used as the
explants. At the time of callus formation, there is some degree of dedifferentiation happens both in
morphology and metabolism. One of the major consequences of this dedifferentiation is that most
plant cultures lose their ability to perform photosynthesis. The necessitates of the addition of other
components such as carbon and vitamins source to the culture media, in addition to the unusual
mineral nutrients.

Materials Required

• Sterile Glass Petri dish

• Sterile forceps
• Sterile scalpel
• Beaker
• Test tube rack
• Waterproof marking pen
• Culture tubes
• Sterile blue cap tubes
• Bunsen burner
• Laminar air flow
• Jar with lid
• Analytical balance

Reagents

• Bleach solution (1.4% of chlorine and wetting agent available)/Soap/Detol

• MS Basal Media -500 ml
• Sterile Media dispense in Tubes (20ml/tube)
• 70% Ethanol
• Sterile distilled water -250 ml
• Healthy, undamaged Explant-such as Carrot/leaf/Dhatura anthers/any other

Reagent preparation:

Stock Preparation
Composition of MS (Murashige and Skoog 1962)

Protocol-
Major salts Concentration mg/L
Ammonium nitrate (NH4NO3) 1,650 mg/l
Calcium chloride (CaCl2 · 2H2O) 440 mg/l
Magnesium sulphate (MgSO4 · 7H2O) 370 mg/l
Monopotassium phosphate (KH2PO4) 170 mg/l
Potassium nitrate (KNO3) 1,900 mg/l.
Minor salts Concentration
Boric acid (H3BO3) 6. 2 mg/l
Cobalt chloride (CoCl2 · 6H2O) 0.025 mg/l
Ferrous sulfate (FeSO4 · 7H2O) 27.8 mg/l
Manganese(II) sulfate (MnSO4 · 4H2O) 22.3 mg/l
Potassium iodide (KI) 0.83 mg/l
Sodium moly date (Na2MoO4 · 2H2O) 0.25 mg/l
Zinc sulfate (ZnSO4·7H2O) 8.6 mg/l
Ethylenediaminetetraacetic acid ferric
sodium (NaFe-EDTA)7.45 mg/L
Copper sulfate (CuSO4 · 5H2O) 0.025 mg/l

Iron Stock Concentration

FeSO4.7H2O 27.8 mg/l
Na2-EDTA 37.8 mg/l
Vitamin Stock Concentration
Myo-Inositol 100 mg/l
Nicotinic Acid 0.5 mg/
Pyridoxine · HCl 0.5 mg/l
Thiamine · HCl 1.0 mg/l
Glycine 2 mg/l
Indole Acetic Acid 5mg/ml stock
Kinetin 5mg/ml stock
BAP 5mg/ml stock
2,4 D 5mg/ml stock

The protocol mentioned above is followed and MS Media was prepared.

1) For 500 ml of MS media preparation, in 350 ml of D/W add 3% sucrose,

myo inositol (100mg/L).
2) Then, add stocks as follows –
1. Macronutrients I – 25ml
2. Micronutrients II – 5ml
3. Iron III – 2.5ml
4. Vitamins IV – 500 μl
3) Add hormone 2, 4 D (1mg/L) – 250 μl
4) Make up the volume up to 450 ml.
5) Adjust pH – 5.5 by using 1N NaOH (only)
6) Again make up the volume to 500ml
7) Then, add (0.9%) agar, Homogenize in the microwave (for 2- 2.5 min x2)
8) Distribute the media in the tube up to 15ml.
9) Cap it, Wrap it and autoclave it for 121°C for 20 minutes.

Result:
Experiment No: ​​ ​ ​ ​ Date:
Callus Induction from explant

The term plant tissue culture (Micro propagation) is generally used for the aseptic culture of cells,
tissues, organs and their components under defined chemical and physical conditions in vitro. The
basic concept of the plant body can be dissected into smaller part termed as “explants” and any
explants can be developed into a whole plant. It is a central innovative areas of applied plant
science, including agriculture and plant biotechnology. This technique is effective because almost
all the plants cell are totipotent; In each cell possesses the genetic information and cellular
machinery necessary to generate the whole organism. Since, this technique can be used to produce
a higher number of plants that are genetically similar to a parent plant as well as to another.
Two concepts, plasticity and totipotency, are the central processes to understand the regeneration
and plant cell culture. Plants, due to its longer life span and sessile nature, have developed a
greater ability to overcome the extreme conditions. Most of the processes inculed in plant
development and the growth, adapt to environmental conditions. When the plant cells and tissues
are cultured in vitro, most of them are generally exhibit a very high degree of plasticity, which
allows one type of organ or tissue to be initiated from another type. Like this way, the whole plant
can be subsequently regenerated. These maintenance of genetic potential is called totipotency.
The plant tissue culture medium is an artificial nutrient supplement of organic and inorganic
nutrients used for cultivation of plant tissue media. The appropriate composition of the medium
largely determines the success of the culture. The culture media used for the in vitro cultivation of
the plant cells are composed of three basic components.
1) Essential elements (normal ions) supplied as a complex mixture of salts.
2) An organic supplements providing vitamins and amino acids.
3) A source of fixed carbon which is usually supplied as sucrose.
When cultured in an appropriate medium having auxin and cytokinin, explants will give rise to an
unorganized, growing and dividing mass of cells called callus. Callus cultures are initiated from a
small part of an organ or tissue segment called the explants on a growth supporting solidified
nutrient medium under sterile conditions. Any part of the plant organ or tissues may be used as the
explants. At the time of callus formation, there is some degree of dedifferentiation happens both in
morphology and metabolism. One of the major consequences of this dedifferentiation is that most
plant cultures lose their ability to perform photosynthesis. The necessitates of the addition of other
components such as carbon and vitamins source to the culture media, in addition to the unusual
mineral nutrients.
Morphology of callus:
Callus varies considerably in appearance and texture, ranging from hard nodular cell masses to
friable soft ones. They maybe white or creamish, orange, green either in whole or part as a result
of chloroplast development. The shape of individual cells within the callus mass ranges from the
near spherical or markedly elongated.
A typical unorganized plant callus initiated from a new explants or piece of previously initiated
calli has three stages of development.
The induction of cell division.
A period of active cell division during which differentiated cells lose specialized features they may
have acquired and become de–differentiated. Cell division usually occurs in the outer layer of
the [Link] when cell division slows down on ceases and when within the callus, there is
increasing cellular differentiat
Callus culturing is performed in the dark while light can be encourage the differentiation of the
callus. At the time of long term culture, the culture may loss the requirement for cytokinin and
auxins. Manipulation of the auxins to cytokinin ratio in the medium can leads to the development
of shoots, roots or somatic embryos from which the plant can be subsequently produced.
Callus culture is useful for many purposes.
1.​ Callus is the starting material for the suspension culture which cells are separated.
2.​ It helps in the production of secondary plant products.
3.​ It is useful for the synthesis of starting compounds that are subsequently modified to yield
the desired product.
4.​ It is the starting materials for vegetative propagation of plants.
Based on the availability of the various invitro techniques, the dramatic increase in their
application to various problems in basic biology, agriculture, horticulture, and forestry. The
applications can divide conveniently into five broad areas;
1. Cell behavior.
2. Plant modification.
3. Germplasm storage and pathogen –free plants.
4. Clonal propagation.
5. Product formation.
6. Improved varities.
Procedure:
1. Wipe down and turn on the laminar air flow 15 minute before doing work in the hood. Flames
–sterilize the instruments.
2. Cut the explant (carrot root/leaf disc/stem) into 3-6 cm long
3. Put the explant section in to a sterile jar having chlorate bleach solution (approximately 1.4%
available chlorine)/Soap solution and shake it for few seconds.
4. Remove the bleach solution into the waste beaker.
5. Cut 1cm of the carrot root section from each end and discard this end portions.
6. Cut 3-5 transverse section (1-5mm thick) across the tap root and transfer each to a fresh sterile
Petri dish with 0.1% Mercuric Chloride solution for 1-2 min.
7. Cut the smaller sections, explants (approximately 5mm square ) from each of the transverse
sections by cutting across the cambium. The following method is recommended
8. Rinse with sterile water to remove excess surface sterilizing agent.
9. Put each explants sections into culture tubes containing the carrot callus initiation medium
(one explants per tube).
10. Seal all the tubes with parafilm to reduce dehydration of the medium.
11. Incubate the culture tubes at 25°C. Examine at weekly intervals and record the changes
observed.
12. Callus formed is removed from the primary explants after 45 days and it is weighed.
13. The calli is subculture into the same medium for further callus growth or to the carrot shoot /
root initiation medium.
Result:
Experiment No: ​ ​ ​ ​ ​ ​ ​ ​ ​ Date:

Isolation of agriculturally important microorganism -Trichoderma spp. From Soil

The genus Trichoderma is a diverse group of free-living fungi in the family Hypocreaceae,
commonly present in all soils. These ascomycetes fungi are opportunistic, avirulent plant
symbionts inhabiting root ecosystems and parasites on other groups of fungi. They reproduce by
chlamydospores and ascospores and proliferate better at mesophilic temperatures (25–35°C) and
wide range of pH. Several findings supported this such as [8] who observed no visible growth of
conidia at 15°C, but retain growth at 25°C and best results at 30°C [9], evaluated the growth of
Trichoderma isolates at different temperatures and pH ranges.

Trichoderma colonizes several ecological niches where they play a vital role; they have been
earlier recognized as effective biocontrol agents of plant-pathogenic fungi, producers of secondary
metabolites of medical importance, and agents of bioremediation. Similarly, their ability to degrade
lignocellulosic biomass to produce second-generation biofuels and other value-added products has
been widely accepted.

Agricultural significance of Trichoderma spp.

Biocontrol potential of the fungus Trichoderma spp. has been recognized. The genes encoding the
enzymes play vital roles in biotic and abiotic stress tolerance, growth of hyphae, degradation of
cell wall, and antagonistic activity against plant pathogens. Trichoderma harzianum and
Trichoderma viride are used as biopesticides and biofertilizers, growth promoters, and inducers of
disease resistance in plants. The former is the main antagonist utilized in management of plant
diseases in agriculture due to its cost-effectiveness and minimal effects on the ecological balance.

Conventional methods for identification of Trichoderma spp. using morphological and cultural
approach have earlier been used. These include arrangement of conidiophores, phialides, and
conidia, while cultural features include linear growth, colony color, growth pattern, and
pigmentation of hyphae. The fungus has revealed different morphologies on various cultivation
media due to genetic factors and environmental and nutritional factors. Green colony pigmentation
after incubation for 7 days at 28°C on potato dextrose agar (PDA) was observed in Trichoderma
cultures isolated from soil samples. Rhizospheric isolates revealed pale or yellowish color of
reverse colonies at 25 and 30°C with rapid growth, loosely arranged conidia, and effused
conidiation.

Trichoderma is efficient in improving vegetative growth of plants and nutrient content of soil
through decomposition and biodegradation. Active substance such as fungal spores is applied as
foliar sprays and pre- and post-planting treatments, during watering and transplanting.
Trichoderma-based products are marketed worldwide and applied in fields, nurseries, and
horticulture for management of fungal soil-borne pathogens such as Pythium and Rhizoctonia. It is
a safe and environmentally friendly method to reduce the detrimental effects of chemical pesticides

Isolation media for Trichoderma

The growth of Trichoderma has been screened on different culture media for various studies using
available, relatively cheaper supporting media such as corn meal agar, oat meal agar, potato
dextrose agar, Czapek’s Dox agar, special nutrient media, carrot agar, rose Bengal agar, selective
media, etc. However, selective media favor growth of Trichoderma strains over other fungi and
hence preferred for easy identification of Trichoderma isolates over rapidly growing fungi that may
overlap it.
Trichoderma selective medium (TSM) is recognized for quantitative isolation of Trichoderma spp.
from soil. It is composed of low glucose level for rapid growth and sporulation of the fungus.
Chloramphenicol is used to inhibit the growth of bacteria, while pentachloronitrobenzene,
p-dimethylaminobenzenediazo sodium sulfonate, and rose bengal are used as selective fungal
inhibitors.
Trichoderma Selective Media (TSM)
0.2 g of MgSO4∙7H2O.
0.9 g of K2HPO4.
0.15 g of KCl.
1.0 g of NH4NO3.
3.0 g of glucose.
0.15 g of rose bengal.
20 g of agar.
0.25 g of chloramphenicol.
0.3 g of p-dimethylaminobenzenediazo sodium sulfonate.
0.2 g of pentachloronitrobenzene.

Recipe is dissolved in 1000 ml distilled water and autoclaved at 121°C, 1.4 kg cm−1 for 15 min.
Then add 0.25 g chloramphenicol and 0.2 g pentachloronitrobenzene into the solution. Keep/store
media at 45°C to prevent solidification.
Method for isolation of Trichoderma
One of the commonest methods reported is the serial dilution of samples. This technique is simple,
cost-effective, and appropriate to handle large samples.
1.​ Soil samples are collected, air dried, and ground into powder.
2.​ 10.0 gm soil sample is serially diluted in sterile saline
3.​ 0.1 ml of each of the prepared dilution is spread evenly on a suitable medium on a petri
dish at 28 ± 1°C for 7 days.
Reference: DOI:10.5772/intechopen.83528
Introductory Chapter: Identification and Isolation of Trichoderma spp.

Observation: Colony characteristics on PDA & TSM Media

Result:
Signature:
Experiment No:​ ​ ​ ​ ​ ​ ​ ​ ​ Date:
Isolation Of N2 Fixing Root Nodule Bacterium Rhizobium From Root Nodules

Introduction
Symbiotic associations between Rhizobium – legume plants are the primary biological contributors
of fixed nitrogen in soil based ecosystem and most studied one also. Symbiotic N2 fixation is
dependent upon the infection of the host root by the appropriate microbial symbiont and the
subsequent development of the required enzymes. Rhizobium is the microsymbiont, which infects
the roots of legume and nodulate. Rhizobium is a common name of the nodulating microsymbiont
which consists of six genera as Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium,
Allorhizobium, Azorhizobium with about 36 species. Rhizobia are aerobic, gram-negative,
non-sporulating rod shaped bacteria which form specialized structures on roots called "nodules".
The size and morphology of the nodules vary with the plant species. The nodules on clover are
relatively small and round or oval shaped. On the other hand, cowpea, common bean, and
soybeans, the nodules are relatively large, round, and firmly attached to the root. On alfalfa, peas
and vetch, the nodules are usually longer and finger-like.
Materials required
Well developed legume nodule
Mercuric chloride (0.1%) and Alcohol (70%)
Forceps, Glass rod , Petri dishes and sterile water blanks

Congo red yeast extract mannitol agar medium

Inoculation needle
Procedure
1.​ Uproot the plant and wash the roots gently and thoroughly under running tap water to
remove soil particles
2.​ Remove the nodules along with root portion without damaging it.
3.​ Immerse intact, undamaged nodules for 5 – 10 seconds in 70 % ethanol
4.​ Rinse the nodules in sterile water
5.​ Surface sterilize the nodules by soaking in 0.1% acidified mercuric chloride or 2.5 – 3.0 %
sodium hypochlorite solution for 1 – 2 minutes
6.​ Wash the nodules in 5 - 6 changes of sterile water using sterile forceps
7.​ Crush the sterilized nodules with a blunt ended sterile glass rod in a large drop of sterile
water in a petri dish / test tube
8.​ Using sterile inoculation needle transfer one loopful of nodule suspension and streak it over
the sterile solidified CRYEMA medium already poured in Petri plate.
9.​ Simultaneously, aliquots of serial dilutions prepared from the nodule suspension may be
used for plating with YEMA either by spread plate method or pour plate method
10.​Incubate the plates at 28°C for 3 – 5 days.
11.​Appearance of circular, raised and white translucent colonies indicates Rhizobium. Red
colored, small colonies are Agrobacterium.

Purification A loopful of Rhizobium colony is taken in the inoculation needle and streaked on
fresh yeast extract mannitol agar plates for purification. The purified cultures of Rhizobia are
maintained on agar slants of the same medium. After isolation, the strains are purified and then
authenticated.

Result:
Experiment No. ​ ​ ​ ​ ​ ​ ​ ​ ​ Date:

Isolation Of N2 Fixing Bacteria - Azotobacter From Soil Samples

Azotobacter Azotobacter is the non-symbiotic, free living, aerobic nitrogen fixing bacterium. In
general, cells are gram negative, polymorphic, form cyst and accumulate polyβ–hydroxybutyrate
(PHB) and produces abundant gum. In addition to N fixation, they secrete plant growth hormones
viz, IAA, GA and growth factors viz., thiamine, riboflavin etc. and produces some antifungal
antibiotics also. Totally six species are identified based on their pigmentation. Among them, A.
chroococcum is the dominant species found in tropical soils.
Isolation Beijerinck was the first to isolate and describe Azotobacter. Azotobacter cells are not
present on the rhizoplane but are abundant in the rhizosphere region. Lack of organic matter in the
soil is a limiting factor for the proliferation of Azotobacter. They depend on the energy derived
from the degradation of plant residues.
Materials Required
Organic matter rich soil sample
Sterile water blanks
Petri plates
Waksman No.77 Medium

Mannitol 10.0 g
CaCO3 5.0 g
K2HPO4 0.5 g
MgSO4.7H2O 0.2 g
NaCl 0.2 g
Ferric chloride Trace
MnSO4.4H2O Trace
N-free washed Agar 15.0 g
pH 7.0
Distilled Water 1000 ml
Waksman No.77 medium

Procedure
1. Weigh one g of sample and put in the 100 ml water blank and mix thoroughly
2. Shake for 15 min for complete dispersion (10-2dilution)
3. Transfer one ml of the suspension to 9 ml water blank (10-3 dilution)
4. Transfer 1 ml of appropriate dilutions (10-3) to Petri dishes
5. Maintain 2 or 3 replications for each dilution
6. Pour melted and cooled media (just before solidification) of about 15 ml and mix well by
shaking clock wise and anti-clock wise for 3 or 4 times and allow it for complete solidification
7. Incubate the plates in inverted position at room temperature for 3-4 days for appearance of
Azotobacter colonies.
8. Determine the moisture content of the soil as described earlier.
Results and Observation Azotobacter produces raised, gummy colonies on agar surface and
aged cultures show yellowish brown/black colouration due to pigment production.
Experiment No:​ ​ ​ ​ ​ ​ ​ ​ Date:
MASS PRODUCTION OF BACTERIAL BIOFERTILIZERS & LIQUID
BIOFERTILIZERS
Introduction
Biofertilizers are defined as preparations containing living cells or latent cells of efficient
strains of microorganisms that help crop plants‟ uptake of nutrients by their interactions in
the rhizosphere when applied through seed or soil. They accelerate certain microbial
processes in the soil which augment the extent of availability of nutrients in a form easily
assimilated by plants. Several microorganisms and their association with crop plants are
being exploited in the production of biofertilizers.N2 fixing organism such as Azospirillum,
Rhizobium, Azotobacter, Gluconacetobacter and PO4 solubilizing bacterial genera
Pseudomonas, Bacillus and PO4 mobilizing Arbuscular mycorrhizal fungi are presently used
as biofertilizers for commercial application .
I. Mass Production of bacterial biofertilizers
Biofertilizers are carrier based preparations containing efficient strain of nitrogen fixing or
phosphate solubilizing microorganisms. Biofertilizers are formulated usually as carrier based
inoculants. The organic carrier materials are more effective for the preparation of bacterial
inoculants. The solid inoculants carry more number of bacterial cells and support the survival
of cells for longer periods of time. The mass production of carrier based bacterial
biofertilizers involves three stages.
1. Mass culturing of microorganisms in fermentor
2. Processing of carrier material

3. Mixing of broth culture with the carrier and packing

1. Mass culturing of Microorganisms in Fermentor Although many bacteria can be used
beneficially as a biofertilizer the technique of mass production is standardized for Rhizobium,
Azospirillum, Azotobacter and phosphobacteria and Gluconacetobacter The media used for
mass culturing are as follows: Rhizobium : Yeast extract mannitol broth. Azospirillum :
Dobereiner's malic acid broth with NH4Cl (1g/lit) Azotobacter : Waksmann No.77 broth
Phosphobacteria : Nutrient broth Gluconacetobacter : LGI broth

appropriate broth in 50 ml flasks and inoculate the mother culture in to the flasks.
 Grow the culture under shaking conditions at 30±2C until maximum cell population of
1010 to 1011 cfu/ml is reached.
 Under optimum conditions this population level could be attained within 4 to 5 days for
Rhizobium; 5 to 7 days for Azospirillum; 2 to 3 days for Phosphobacteria and 6-7 days for
Azotobacter & Gluconacetobacter. The culture obtained in the flask is called starter culture.
 Use the starter to inoculate the broth in large size flasks of 250 ml, 500 ml, 3 liters and 5
liters and grow until required level of cell count is reached.
 For large scale production of inoculant, inoculum from starter culture is transferred to
large flasks/seed tank fermentor.
Fermentor Bacterial biofertilizers are normally mass cultured in fermenters. Fermentor is the
vessel which maintains the controlled environmental conditions for the growth of
microorganisms and provides access for inoculation, sampling, aeration and cleaning. It
should be made of stainless steel to withstand high pressure and also to resist corrosion. High
quality fermentor will have smooth surface inside.
Basic functions of a fermentor
 To provide a controlled environment for the growth of microorganisms in order to obtain a
desired product.

 Fermentor vessel should be capable of being operated aseptically for a number of days.

 Should withstand high pressure. The power consumption should be as low as possible.
The

vessel should be designed to require minimal use of labour for operation.

 The vessel should be constructed to ensure smooth internal surfaces without cracks and
crevices.

Sterilization of growth medium in the Fermentor

 Prepare required quantity of growth medium and adjust to the required pH
 Pour the medium into the fermentor vessel after closing the sampling valve
 Keep the air outlet valve open

Bring the growth medium to boiling under maximum heat by using steam generator
 Close the air outlet valve and allow the pressure to build up inside the vessel
 Maintain a pressure of 15 1b / in2 at 121°C for 20 minutes
 Switch off the fermentor and cool the medium by circulating cool water.
Mass culturing in Fermentor
 Spray the inoculation port with alcohol and flame thoroughly
 Allow the port to cool, inoculate the media in the fermentor vessel with the log phase
culture grown in 5 litre flask. Usually 1 -2 % inoculum is sufficient, however inoculation is
done up to 5% depending on the growth of the culture in the larger flasks.
 Turn on the air pump, open the air outlet valve
 Regulate the air flow to 3-10 lit of air per hour per lit. of the medium. The sterile air
provides aeration as well as agitation for the growth of culture
 Draw samples and analyze for growth, periodically if necessary
 Once the culture reaches full growth turn off the air supply and harvest the broth with the
population load of 109 cells ml-1 after incubation period through the sampling port.
 There should not be any fungal or any other bacterial contamination at 10-6 dilution level
 It is not advisable to store the broth after fermentation for periods longer than 24 hours.
Even at 4oC number of viable cells begins to decrease.

2. Processing of carrier material The use of ideal carrier material is necessary in the
production of good quality biofertilizer. Peat soil, lignite, vermiculite, charcoal, press mud,
farmyard manure and soil mixture can be used as carrier materials. The neutralized peat
soil/lignite are found to be better carrier materials for biofertilizer production.
Characteristics of an Ideal carrier
 Cheaper in cost

 Should be locally available

 High organic matter content

 Should not be toxic

 Water holding capacity of more than 50%

 Easy to process, friability and vulnerability.

 Amenable for mixing

Preparation of carrier material:

 Powder the carrier material (peat or lignite) to a fine powder so as to pass through 212 
IS sieve.

 Neutralize the pH of the carrier material with the help of calcium carbonate (1:10 ratio) ,
since the peat soil / lignite are acidic in nature ( pH of 4 - 5) Sterilize the neutralized carrier
material in an autoclave to eliminate the contaminants. For large scale production gamma
Specification of the polythene bags
 The polythene bags should be of low density grade.
 The thickness of the bag should be around 50 – 75 micron.
 Each packet should be marked with the name of the manufacturer, name of the product, strain
number, the crop to which recommended, method of inoculation, date of manufacture, batch number,
date of expiry, price, full address of the manufacturer and storage instructions etc.,

Storage
 The packet should be stored in a cool place away from the heat or direct sunlight.
 The packets may be stored at room temperature or in cold storage conditions in lots in plastic
crates or polythene / gunny bags.

Product specifications
 There should be more than 108 cells / g of inoculant at the time of preparation and107 cells/ g on
dry weight basis before expiry date.
 It should not have any contaminant at 10 -5 dilution

General procedure for the production of carrier based inoculants

 Prepare the starter culture in small size flasks
 Prepare the seed culture in large size flasks or in seed tank fermentor
 Inoculate the seed culture into the fermentor @ 1-3 % and multiply the culture in fermentor for
large scale production with the respective growth medium to the population level of 109 cells/ml broth
 Mix the broth with prepared carrier material (neutralized and should pass through 212 micron IS
sieve) to 40-50% moisture level
 Pack it with low density printed poly bags (50-75 micron)
 The final product should contain the population load of 108 cells/g product at preparation

II. Mass production of liquid biofertilizers All the bacterial biofertilizers except Rhizobium are
produced in liquid formulations also. Liquid biofertilizers are produced through three step process.
1. Preparation of starter culture and seed culture

Prepare the starter culture from the mother culture in the respective growth medium as given for
carried based inoculants 2. Mass culturing in fermentor
 Do the mass culturing similar to carried based inoculants in the fermentor.
irradiation and sun drying method is followed.

Schematic representation of mass production of bacterial biofertilizers

Mixing of broth culture with the carrier and packing Add the bacterial culture drawn
from the fermentor to the neutralized and sterilized carrier material to the moisture content of
35 to 45% on wet basis. The carrier and broth can be mixed either manually (by wearing
sterile gloves) or mechanically. After mixing, pack the inoculants in 200 g quantities in
polythene bags, seal with electric sealer and allow for curing for 2 -3 days at room
temperature (curing can be done by spreading the inoculant on a clean floor/polythene sheet/
by keeping in open shallow tubs/ trays with polythene covering for 2 -3 days at room
temperature before packaging). Curing improves the cell count to 109 to 1010 cells /g. After
curing it is then packed in low density polythene bags. Theinoculants may be allowed for
curing even after packing for 3- 4days at room temperature.
Specification of the polythene bags
 The polythene bags should be of low density grade.
 The thickness of the bag should be around 50 – 75 micron.
 Each packet should be marked with the name of the manufacturer, name of the product,
strain number, the crop to which recommended, method of inoculation, date of manufacture,
batch number, date of expiry, price, full address of the manufacturer and storage instructions
etc.,

Storage
 The packet should be stored in a cool place away from the heat or direct sunlight.
 The packets may be stored at room temperature or in cold storage conditions in lots in
plastic crates or polythene / gunny bags.

Product specifications
 There should be more than 108 cells / g of inoculant at the time of preparation and107
cells/ g on dry weight basis before expiry date.

 It should not have any contaminant at 10 -5 dilution

General procedure for the production of carrier based inoculants

 Prepare the starter culture in small size flasks

 Prepare the seed culture in large size flasks or in seed tank fermentor

 Inoculate the seed culture into the fermentor @ 1-3 % and multiply the culture in
fermentor for large scale production with the respective growth medium to the population
level of 109 cells/ml broth

 Mix the broth with prepared carrier material (neutralized and should pass through 212
micron IS sieve) to 40-50% moisture level

 Pack it with low density printed poly bags (50-75 micron)

 The final product should contain the population load of 108 cells/g product at preparation

II. Mass production of liquid biofertilizers All the bacterial biofertilizers except Rhizobium
are produced in liquid formulations also. Liquid biofertilizers are produced through three step
process.
1. Preparation of starter culture and seed culture

Prepare the starter culture from the mother culture in the respective growth medium as given
for carried based inoculants
2. Mass culturing in fermentor
 Do the mass culturing similar to carried based inoculants in the fermentor.
Harvest the broth once the population reaches the cell load of 10 10 cell per ml
broth
2. Preparation of liquid formulation
 Fill the harvested culture in the sterile plastic container of one liter or 500 ml
capacity
 Add Glycerol @ of one ml per liter broth to arrest the metabolic activities of the
cell so as to avoid bursting of the container under storage
 Seal the mouth with sterile caps and store under room temperature
TESTING QUALITY CONTROL OF BIOFERTILIZERS
Quality control must begin with the maintenance of mother culture and broth
culture before addition to the carrier and finished product [Link] inoculant
(1) Mother culture Repeated sub-culturing or longer period of storage may result
in the loss of nodulation ability of Rhizobial isolates. Plant infection test is
conducted by the seedling agar tube, Leonard jar assembly or by paper towel
method to check the nodulation ability. (2) Broth culture Check for contamination
by (a) Streaking on glucose peptone agar plates. Slow growth or no growth ensures
Rhizobium (b) Performing Gram staining – absence of gram positive cells ensures
purity of the culture (3) Inoculant Check the population level and presence of
contaminants by serial dilution plate technique using yeast extract mannitol congo
red agar medium (CRYEMA). (4) BIS (Bureau of Indian Standards) specifications
for Rhizobium inoculant
Inoculation should contain a minimum of 108 viable Rhizobium cells/g of the
carrier on dry weight basis at the time of manufacture and 107 cells on expiry date
marked on the pocket.
No contaminants at 106 dilution
pH of inoculant should be 6.0 to 7.5
Carrier material should pass through 106 micron size sieve
Packing low density polythene bags of 50 - 75 μ
Each packet should be marked with the details – Name of the product,
leguminous crop for which intended, strain number, date of manufacture, date of
expiry, method of application and storage instructions.
B. Azospirillum inoculant The Azospirillum inoculant packets may be subjected to
the quality control test-during preparation as well as during storage. The quality of
the Azospirillum inoculant may be tested by the following methods.
Determination of Azospirillum population in inoculant by MPN technique
2. Checking the presence of contaminating bacteria in the inoculant by serial
dilution technique using nutrient agar medium.
Quality control specification for Azospirillum inoculant
Azospirillum inoculant should contain a minimum of 109 viable Azospirillum
cells/g of the dry carrier at the time of manufacture and 107 cells/g dry carrier at
15 days before the expiry date mentioned on the packets
No contamination with other microorganisms
pH of inoculant should be 7.2-7.5
Carrier material should be neutral and pass through 100 micron sieve
Each packet should be marked with strain number, date of manufacture, date of
expiry, method of application etc.
A. Phosphobacterial inoculant
The quality control specifications prescribed for Azospirillum inoculant is followed
for phosphobacterial inoculant.
Inference:
BIS STANDARDS FOR ASSESSING THE QUALITY OF BIOFERTILIZERS