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GONDWANA UNIVERSITY, GADCHIROLI

FACULTY OF SCIENCE AND TECHNOLOGY

PRACTICAL MANUAL

For

T. Y. B. Sc.

BOTANY

SEMESTER - VI

Under

Choice Based Credit System (CBCS)

Discipline Specific Elective-II

Plant Biotechnology

Prepared by

Dr. Mrs. Vasanti Rewatkar

Shri Dnyanesh Mahavidyalaya, Nawargaon

Dist. Chandrapur

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SYLLABUS FOR

B. Sc. Semester VI (C.B.C.S.)

Discipline Specific Elective-II (DSE-II)

PAPER-I

(Plant Biotechnology- I)

UNIT – I (12 Periods)

Plant Tissue Culture:

Definition, Historical perspective, Composition of media; nutrient and hormone requirements (role of

vitamins and hormones), Major types of tissue culture media (MS, B5, N6).

UNIT – II (12 Periods)

Basis of PTC techniques:

Totipotency, differentiation, dedifferentiation, redifferentiation, regeneration (direct and indirect),

organogenesis, embryogenesis (somatic and zygotic).

UNIT – III (12 Periods)

Tissue Culture Techniques:

Micropropagation, virus elimination, protoplast isolation, culture and fusion, Secondary

metabolite production.

UNIT – IV (12 Periods)

Tissue Culture Techniques:

Anther culture, pollen culture, ovary culture, production of haploids, triploids and hybrids, hardening

of the tissue culture raised plants for field plantation, cryopreservation, germplasm conservation.

Laboratory Exercises:

1. To study laboratory setup for Plant Tissue Culture techniques.

2. To get acquainted with equipment’s and instruments used in Plant Biotechnology laboratory.

3. To study various sterilization methods employed in Plant Biotechnology Laboratory.

4. To prepare Murashige and Skoog plant tissue culture medium.

5. To conduct surface sterilization and inoculation of different explants (leaf, nodal buds, seeds)

from suitable plant for callus induction.

6. To demonstrate anther culture.

Suggested Readings:

1. Biotechnology Expanding Horizons: B. D. Singh.

2. Comprehensive Biotechnology: K. G. Ramawat and Shaily Goyal.

3. Biotechnology: U. Satyanarayana.

4. Plant Cell and Tissue Culture: Indra K. Vasil.

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PAPER-II

(Plant Biotechnology- II)

UNIT – I (12 Periods)

Transgenic Crop Production:

Definition, methods of gene transfer – direct (electroporation, microinjection, microprojectile

bombardment, silicon carbide mediated).

UNIT – II (12 Periods)

Transgenic Crop Production:

Indirect method of gene transfer - Agrobacterium-mediated gene transfer,

Selection of transgenics – selectable marker and reporter genes (luciferase, GUS, GFP).

UNIT – III (12 Periods)

Applications of Plant Biotechnology:

Pest resistant plants (Bt-cotton); herbicide resistant plants (Roundup Ready soybean); transgenic

crops with improved quality traits (Golden rice); improved horticultural varieties (Moondust

carnations).

UNIT – IV (12 Periods)

Applications of Plant Biotechnology:

Transgenic plants producing edible vaccines, biodegradable plastic, chloroplast transformation;

bio safety concerns and ethics.

Laboratory Exercises:

1. To demonstrate micropropagation from callus/explants.

2. To isolate and culture protoplasts.

3. To prepare suspension culture for secondary metabolite production.

4. To study different methods of direct gene transfer using models / charts / photographs / ppt

/ video.

5. To study steps involved in the production of Bt cotton / Golden rice through models / chart

/ photographs / ppt / video.

6. To study steps involved in the production of transgenic plants producing edible vaccine /

biodegradable plastic using models / charts / photographs / ppt / video.

Suggested Readings:

1. Biotechnology Expanding Horizons: B. D. Singh.

2. Comprehensive Biotechnology: K. G. Ramawat and Shaily Goyal.

3. Biotechnology: U. Satyanarayana.

4. Plant Cell and Tissue Culture: Indra K. Vasil.

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GONDWANA UNIVERSITY, GADCHIROLI

Practical Question Paper Pattern

B.Sc. BOTANY CBCS

SEMESTER – VI

PRACTICAL

(Based on DSE theory papers - Plant biotechnology)

[Time 5 Hours] [Max. Marks – 30]

Que. 1: Based on Paper I 4Marks

Que. 2: Based on Paper I 4Marks

Que. 3: Based on Paper II 4 Marks

Que. 4: Based on Paper II 4 Marks

Que. 5: Spotting 6 Marks

1. Based on Paper I

2. Based on Paper I

3. Based on Paper I

4. Based on Paper II

5. Based on Paper II

6. Based on Paper II

Que. 6: Viva-voce 3 Marks

Que.7: Practical Record and Excursion Report 5 Marks

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B.Sc. TY/ Semester – VI

Practical experiments & spotting to be conducted

PAPER – I 1. To prepare Murashige and Skoog plant tissue culture medium.

2. To study various sterilization methods employed in Plant Biotechnology

Laboratory.

3. To conduct surface sterilization and inoculation of different explants (leaf,

nodal buds, seeds), from suitable plant for callus induction.

4. To demonstrate anther culture.

The equipment’s and instruments used in Plant Biotechnology laboratory.

SPOTTING:

Autoclave Hot

air oven

Laminar air flow (LAF)

pH meter

Electronic balance

Scalpel with blade / surgical scalpel

Spatula

Scissors

Arrow headed sharp needle Laboratory

Spirit

Sodium hypochlorite

Nutrient medium Agar

Callus

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PAPER – II

1. To demonstrate micropropagation from callus/explants.

2. To isolate and culture protoplasts.

3. To study different Methods of direct gene transfer.

4. Steps involved in the production of Bt cotton.

5. Steps involved in the production of Golden rice.

6. Steps involved in the production of transgenic plants producing edible

vaccine / biodegradable plastic.

SPOTTING: Photographs/ models/ charts

Gene gun

Micro pipette

Electrophoration

Agro bacterium

Bt cotton Golden

rice

Moon dust carnation

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EXPERIMENT NO.1 (PAPER - I):

Aim: To prepare Murashige and Skoog plant tissue culture medium.

Requirements:

Murashige and Skoog (MS) medium with vitamins; plant growth hormones like NAA

(1-naphthaleneacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid); BAP (Benzyl

amino purine); Sucrose; NaOH; KOH; Agar; NaCl; HCl; Petri dish; Pipette;

Parafilm; Weighing box; Aluminium foil; Autoclave; Spatula; Balance; Sterile

containers for stock solutions (Schott bottle or universal bottle); Measuring cylinder

(1L and 100 ml); Magnetic stirrer and bar; pH meter; Erlenmeyer flasks (250 ml, 500

ml);.

Principle:

Solid and liquid media for plant cultures can be prepared either by dissolving the

commercially available basal salts mix or by using self-prepared media. Alternatively,

macronutrient and micronutrient stock solutions are prepared and stored at 40C until

used. Organic compounds are recommended to be stored not more than 2 weeks in

solution. Heat-labile compounds such as plant growth regulators (PGRs), antibiotics

or glutamine should be filter sterilized and added when the medium has been

autoclaved and cooled.

Procedure:

A. Preparation of Plant Growth Regulator Stock Solutions:

All stock solutions are prepared 1000 times more concentrated than the

concentration to be used in the medium.

For example, 0.5 mg/L of 2, 4-D is added to the basal medium. Then the

stock solution is prepared with a concentration of 500 mg/L.

1. Add 5 mg or 0.5 g plant growth regulator (PGR) into 50 ml distilled water.

2. Add 2-5 ml solvent (1 M NaOH) to dissolve the powder.

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3. Stir the solution with magnetic stirrer while adding water to bring the final volume

to 100 ml.

4. Filter each stock solution using 0.22 μm syringe filter and transfer them

into sterile containers before storing them in 4C. The stock solution can last up to 3

months.

Volume of Stock solution = Desired PGR concentration X Medium volume

Stock solution concentration

Therefore, to prepare 1 liter of medium, the amount of PGR required are as given in

Table 1.

Adding 1.0 ml of stock solution to 1 L medium gives 0.05 mg/L concentration of PGR

in the medium.

B. Preparation of Liquid and Solid Media Using Commercially Available Basal

Salts Mix (1L Medium)

1. Measure 1L of distilled water in the measuring flask.

2. Weight the correct amount of MS with vitamins powdered medium. Dissolve it in

800 ml of distilled water.

3. Add 1 ml of the required PGRs. Stir the mixture well.

4. Add 30 g sucrose while continuously stirring the mixture. Add more distilled

water to make up 950 ml volume.

5. Adjust the pH of medium to 5.7 with 1.0 M NaOH (for preparing solid media,

agar is added to the medium at this stage by constant stirring with slow heat to the

boiling point to make sure that the agar is fully dissolved).

6. Add distilled water to make a final volume of 1L.

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7. Transfer and distribute the liquid media into small flasks (250 ml), while the

medium with agar is transfer into a Schott bottle.

8. Cover the flasks with sponge and aluminium foil and close the culture vessels with

parafilm.

9. Autoclave at 121C, 15 psi for 20 minutes. 10. Test the sterility of media by

leaving them at 25C for 4 days to observe any contamination.

For preparing solid media:

1. Cool down the medium after sterilisation to about 40C.

2. Pour the medium onto petri dishes under the laminar flow cabinet and leave the

medium to solidify.

3. Keep the petri dishes upside down to avoid contamination by evaporation

and condensation if any. For long period of storage, keep them in a cool and dry

place.

Medium Components (mg/L) Murashige and Skoog

Macronutrients

Ammonium Nitrate (NH4NO3) 1650.0

Potassium nitrate (KNO3) 1900.0

Calcium Chloride (CaCl2 2H2O) 440.0

Magnesium Sulphate (MgSO4 7H2O) 370.0

Potassium bi Phosphate (KH2PO4) 170.0

Sodium phosphate monobasic

(NaH2PO4 H2O)

--

Calcium nitrate (CaNO3 4H2O) --

Sodium Sulphate (Na2SO4) --

Potassium Chloride (KCl) --

Micronutrients

Potassium iodide (KI) 0.83

Boric acid (H3BO3 ) 6.2

Manganese sulphate (MnSO4 .4H2O) 22.3

Zinc sulphate (ZnSO4.7H2O ) 8.6

Sodium Molybdate (Na2MoO4 . 2H2O) 0.25

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Copper sulphate (CuSO4 .5H2O) 0.025

Cobalt Chloride (CoCl2 .6H2O) 0.025

Na2EDTA 37.3

Ferrous sulphate (FeSO4 . 7H2O) 27.8

Molybdenum trioxide

Vitamins and organic supplements

Myo-Inositol 100.0

Glycine 2.0

Thiamine HCl 0.1

Pyridoxine HCl 0.5

Nicotinic acid 0.5

Sucrose 30,000

Glycine (free base) 2.0

Others

Agar – agar powder (solidifying agent) 10,000

GrowthHormones

Naphthalene Acetic Acid (Auxin)

Benzyle Amino Purine (Cytokinin)

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EXPERIMENT NO.2 (PAPER - I):

Aim: To study various sterilization methods employed in Plant Biotechnology

Laboratory.

Requirements: Autoclave; Hot air oven; Sodium hypochlorite, Laboratory spirit.

Principle: The process of making something free from bacteria or other living

microorganism and pathogen is called 'Sterilization". Since artificial and readymade

food is available in the culture media, there is a chance for the growth of

microorganisms. So, it is necessary to sterilize the instruments and equipments used

in plant tissue culture. Even the nutrient media has to be sterilized. The methods of

sterilization differs with the material used in plant tissue culture.

Methods:

1. Autoclaving: It is also called wet heat. Pressurized steam is used to heat the

material to be sterilized. This is very effective method to kill all microbes, theirspores

and also viruses. Autoclaving kills microbes by hydrolysis and coagulation of cellular

proteins. Intense heat comes from pressurized heat, which has very high latent heat.

Usually, plastic ware, metal ware with plastic handles, rubber gloves are sterilized by

using autoclave. The nutrient medium is sterilized in an autoclave, but before adding

heat sensitive vitamins and plant growth hormones.

2. Hot air sterilization: Glassware like culture vials, test tubes, glass petri-dishes,

pipettes and metal instruments and aluminum foil can be sterilized by exposure to hot

dry air (about 160-1800C) for 2-4 hours in the hot air oven.

3. Flaming or Baking: This is also called Dry heating. Either direct or indirect flames

or hot fumes are used for sterilizing the glassware and other heat resistant materials

and utensils that are used in plant tissue culture. Dry heat tends to kill microbes by

oxidation of cellular components. Hot air oven is used for sterilization by dry heating.

4. Chemical sterilization: In plant tissue culture particularly the explants are surface

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sterilized using certain disinfectant chemicals like Sodium hypochlorite, calcium

hypochlorite, hydrogen peroxide, silver nitrate, and Mercuric chloride. Commercial

Bleach contains about 5% sodium hypochlorite, and so it may be used at a

concentration of 10-20 % which is equivalent to 0.5 - 1.0% of Sodium hypochlorite. It

is beneficial to keep the materials, that are to be sterilized, in 70% ethyl alcohol

before treating with disinfectant chemicals.

If the explant is a seed, then it will be better to add few drops of TWEEN 20, a

wetting agent, to the disinfectant solution. It reduces the surface tension and allows

better surface contact.

5. Filter sterilization is used when the nutrient media contains heat and hormones that

may decompose (become useless) during autoclaving. So, the solutions with these

compounds require filter sterilization. The membrane filtration traps contaminants that

are larger than the pore size on the surface of filter membrane. A filter of differing pore

sizes can be added in series for better separation of multiple microorganisms form the

culture media.

6. Radiation: Electromagnetic radiation like UV rays and X - rays are excellent tools

for sterilizing, as they have damaging effect on the DNA. UV lamps are used to

sterilize the culture room walls and ceiling areas. While culture room floor can be

sterilized using simple commercially available disinfectant solutions in the market

like dettol, phenyl, Lizol etc…

…………………………..XXX…………………………………….

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EXPERIMENT NO.3 (PAPER - I):

Aim: To conduct surface sterilization and inoculation of different explants (leaf,

nodal buds, seeds), from suitable plant for callus induction.

Requirements: Explant (leaf or nodal bud or seed), Teepol, Sodium hypochlorite,

mercuric chloride, Petridishes, surgical blade or scalpel with blade, forceps, ethyl

alcohol, laboratory spirit.

Principle: The process of making something free from bacteria or other living

microorganism and pathogen is called 'Sterilization". Since artificial and readymade

food is available in the culture media, there is a chance for the growth of

microorganisms. So, it is necessary to sterilize the explants. Plant material which is to

be cultured, should be surface sterilized to remove the surface borne microorganisms.

Procedure:

A. Surface sterilization of explant:

1. Take the dry seeds or Cut the explant (leaf or nodal bud) with a surgical blade or

sterile scalpel.

2. Thoroughly washed plant material in tap water, is immersed in 5% v/v solution

of liquid detergent such as 5% ‘Teepol’ solution for 10-15 minutes. Then wash

again the explant thoroughly in tap water and finally in distilled water. This step

can be done in the general laboratory.

3. Subsequent steps are done in front of a laminar air flow or the pre-sterilized

inoculation chamber. Now, Dip the explants in 70% ethyl alcohol for 1 Min.

4. Immediately transfer the material into a sterile bottle and pour 0.1% mercuric

chloride (HgCl2) or 5-10% Sodium hypochlorite (v/v) solution and keep for 10

-15 minutes. The bottle is frequently swirled for shaking so that all surfaces of

the plant material come equally in contact with the disinfectant.

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5. Wash the explants thoroughly with several changes of autoclaved distilled water

to remove all traces of sterilant. Now, the explants are ready for culture.

B. Inoculation of Explant:

Successful control of contamination largely depends upon the precautions

taken to prevent the entry of microorganisms at the time of transferring the sterilized

explants on the nutrient medium. Dust, hair, hands and clothes are potential sources

of contamination. The inoculating chamber should be dust free and the operator

should wear sterile headgear and aprons before entering the culture area.

The hands should be wiped with 95% alcohol and the transfer area also should

be cleaned and wiped with 95% alcohol before starting the transfer process. Talking

or sneezing should be avoided during transfer of explant into the media. The neck or

mouth of culture container should be flamed, the transferring instruments also to be

flamed and dipped in alcohol.

Care should be taken so that the explant should not touch the edge of culture

vessel, and after transferring the mouth of culture vessel should be closed by cap or

by cotton plug and the petridishes are to be sealed by ‘Parafilm’. During the transfer,

it is also necessary to ensure that the plant tissue is exposed to the media properly.

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EXPERIMENT NO.4 (PAPER - I):

Aim: To demonstrate anther culture.

Requirements: Anthers (of any suitable plant), MS medium (commercially available

powder can be used), Agar powder, Sucrose, Sodium hypochlorite, mercuric chloride,

activated charcoal, petridishes, surgical blade or scalpel with blade, forceps, ethyl

alcohol, laboratory spirit, Beakers, culture vessels and laminar air flow chamber.

Principle: Anther culture is a technique where anthers are cultured in vitro in a

nutrient medium under controlled conditions. It produces haploid male plants. The

process of anther culture is called "Androgenesis". Age of the plant and maturity

stage of the anther influences anther culture.

Procedure:

1. Collect disease free anthers from unopened flower buds. Keep them in petridish

and clean gently with tap water to remove surface dust.

2. Transfer the selected buds to the laminar air flow chamber.

3. Surface sterilize the buds with 70% ethyl alcohol for 10 seconds and then

sterilize the buds with 20% sodium hypochlorite for 10 minutes.

4. Wash buds three times, with distilled water and transfer them to sterilized

petriplate.

5. Then with the help of a scalpel, separate the stamen from the bud and remove

the filaments from the anther.

6. Now, transfer an anther onto the solid or liquid nutrient medium and incubate it

for 3-4 weeks at 24-28 0C temperature in dark.

7. Then, either by embryogenesis or organogenesis, haploid plantlets would

appear in 3-4 weeks.

8. During this stage, incubate the culture at 24-28 0C for 12-18 hours in light

and for 6-12 hours in the dark.

9. The plantlets of about 50mm tall are transferred to the pot containing bio

compost. Then a sterilized glass beaker is used to cover the pot.

10. Remove glass beaker after some weeks and transfer the plant to a large pot.

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Callus initiation from anthers of Bignonia grandiflora (courtesy-: from Dr.Malesh Reddy)

Callus

Anther

Culture media

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EXPERIMENT NO.5 (PAPER - II):

Aim: To demonstrate micro propagation from callus/explants.

Requirements: Suitable explant, MS medium powder (commercially available), Agar

powder, Sucrose, Plant growth hormones like NAA, BAP, 0.1% Tween 20, 5- 10%

Sodium hypochlorite, 1% mercuric chloride, activated charcoal, Petridishes, surgical

blade, scalpel with blade, forceps, ethyl alcohol, laboratory spirit, Beakers and

laminar air flow chamber.

Principle: Use of small pieces of plant tissues or organs grown in vitro (under

controlled conditions) to produce large number of plants is called

“Micropropagation”. Millions of plants can be produced in short time period.

Selection of the mother plant or explant is the key step in Micropropagation. Callus is

produced using basal nutrient medium, while complete plantlets are produced when

standard medium containing plant growth hormones is used. Usually

micropropagation is done for economically important plant species.

Procedure:

1. Select the suitable virus or disease free mother plant for Micropropagation.

Collect small shoots or axillary buds as explants.

2. Clean the explants with tap water to remove dust. Add 0.1% Tween 20 to water

to remove other contaminants.

3. Dip the explants in 70% ethyl alcohol for 60 seconds.

4. Surface sterilize the explants with 5-10% Sodium hypochlorite for 10 minutes.

The bottle is frequently swirled for shaking so that all surfaces of plant material

come equally in contact with disinfectant.

5. Then wash the explants with culture grade distilled water thoroughly to remove

excess sterilant.

6. Prepare MS medium with vitamins from commercially available basal salt mix.

Add 1 ml of the required PGRs (plant growth regulators). Stir the mixture well.

7. Add 30 g sucrose while continuously stirring the mixture. Add more distilled

water to make up 950 ml volume.

8. Adjust the pH of medium to 5.7 using 1.0 M NaOH. Make the final

volume to 1L.

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9. Add agar to the medium at this stage by constant stirring with slow heat to the

boiling point to make sure that the agar is fully dissolved. Agar makes the

medium solid.

10. Transfer and distribute the liquid media into small flasks (250 ml), while the

medium with agar is transfer into a Schott bottle.

11. Cover the flasks with sponge and aluminium foil.

12. Autoclave the medium at 1210C, 15 psi for 20 minutes. Test the sterility of

media by leaving them at 250C for 4 days to observe any contamination.

13. Now, transfer the explants to culture vessels with the help of sterile forceps,

and close the culture vessels with parafilm.

14. After few days callus can be observed when basal medium is used. Then the

callus can be sub-cultured to making millions of plants through

Micropropagation.

15. Using plant growth hormones plantlets develop in the culture media.

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EXPERIMENT NO.6 (PAPER - II):

Aim: To isolate and culture protoplasts.

Requirements: Suitable explant (protoplast of any cell), MS medium powder

(commercially available), Sucrose, Agar powder, Cellulase, pectinase, Plant growth

hormones like NAA, BAP, 0.1% Tween 20, 5-10% Sodium hypochlorite, NaOH,

Petridishes, surgical blade, scalpel with blade, forceps, ethyl alcohol, laboratory spirit,

Beakers and laminar air flow chamber.

Principle: The plant cells without walls are called protoplasts. To obtain protoplasts,

the cell walls are to be dissolved aseptically. The protoplasts can be cultured in the

nutrient medium to re grow into plantlets. Young leaves of healthy plants are most

suitable material for isolation of protoplasts.

Procedure:

A. Isolation of protoplasts:

1. Select a young leaf from a healthy plant.

2. Cut the leaf with sterilized scalpel or surgical blade.

3. Wash the leaf with tap water to remove any dust. Then clean it with surface

disinfectant like 0.1% Tween 20.

4. Keep the leaf in a petri dish having 5% sodium hypochlorite and wash

thoroughly for 10 minutes.

5. Then wash the explants with culture grade distilled water thoroughly to remove

excess sterilant.

6. Remove the Lower epidermis with the help of forceps. Keep the peeled leaf

segments in cell wall degrading enzyme mix (cellulase + pectinase) for a

specific time and protoplasts are isolated.

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B. Culturing protoplasts:

1. Prepare MS medium with vitamins from commercially available basal salt mix.

Add 1 ml of the required PGRs (plant growth regulators). Stir the mixture well.

2. Add 30 g sucrose while continuously stirring the mixture. Add more distilled

water to make up 950 ml volume.

3. Adjust the pH of medium to 5.7 using 1.0 M NaOH . Make the final volume

to 1L.

4. Add agar to the medium at this stage by constant stirring with slow heat to the

boiling point to make sure that the agar is fully dissolved. Agar makes the

medium solid.

5. Transfer and distribute the liquid media into small flasks (250 ml), while the

medium with agar is transfer into a Schott bottle.

6. Cover the flasks with sponge and aluminium foil.

7. Autoclave the medium at 1210C, 15 psi for 20 minutes. Test the sterility of

media by leaving them at 250C for 4 days to observe any contamination.

8. Now, transfer the explants to culture vessels with the help of sterile forceps and

close the culture vessels with parafilm.

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EXPERIMENT NO.7 (PAPER - II):

Aim: To study different methods of direct gene transfer.

Principle: The phenomenon of introduction of exogenous DNA into the genome to

create and maintain a stable and heritable character is called ‘Transgenesis’. The

foreign gene or genetic material that has been transferred naturally or by genetic

engineering technique from one organism to another is called ‘Transgene’.

Gene transfer is a technique to efficiently introduce foreign gene(s) into the

genome of target plants or cells and make them stable, so that they are inherited to

give desired results. Gene transfer is useful in creating new genetically modified

plants. The direct transfer relies on the delivery of large amounts of DNA. Usually the

plant cells are not permeable to any foreign material. So, the cells are transiently

permeabilized through Particle bombardment, Electroporation, ultrasound, use of

chemicals etc. This direct method is mostly used for gene transfer in cereal crops and

some dicotyledonous crops.

Methods:

1. Particle bombardment is a physical method used to introduce foreign DNA into

plant cells. It is the most effective method in direct gene transfer technique.

2. The DNA (to be transformed), is coated onto microscopic beads which are

made up of either gold or tungsten.

3. The coated particles are loaded into a particle gun or gene gun and accelerated

to high speed either by the electrostatic energy released from a droplet of water

exposed to high voltage or using pressurized helium gas; the target could be

plant cell suspensions, callus cultures, or tissues.

4. As the microprojectiles enter the cells, transgenes are released from the particle

surface for subsequent incorporation into the plant’s genome (DNA).

5. This method has been used to produce stable transformed cell lines and plants

of several species. For example wheat, rice, maize and tobacco.

6. Electroporation: A foreign DNA is transferred or introduced into the plant cell

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by exposing them for a very brief period of high voltage electric pulses. These

pulses induces transient (short-lived) pores in plasma lemma. This process is

called 'Electroporation'.

7. Electroporation is done by using either (a) Low voltage pulses for long time -

300-400 V/cm for 10-50 milliseconds, or (b) High voltage pulses for short

period - 1000-1500 V/cm for 10 micro seconds. This method is more stable.

8. Micro injection is another method for DNA transfer to cereal plants.

9. This method is used to transfer DNA into large cells using a specialized optical

microscope setup which is commonly called as 'micromanipulator'.

10. This process is frequently used to insert genetic material into a single cell. Normally the DNA is

injected into the nucleus of plant cell.

11. The DNA coated silicon carbide fibres are vortexed with ‘plant material

(suspension culture, calluses). During the mixing, DNA adhering to the fibres

enters the cells and gets stably integrated with the host genome.

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12. PEG method is used for protoplasts only. Poly Ethylene Glycol commonly

called as PEG stimulates endocytosis and therefore DNA uptake occurs easily.

The protoplasts are kept in the 20% PEG solution.

13. Intact surviving protoplasts are then cultured to form cells with walls and then

they form colonies. Plant protoplasts treated with polyethylene glycol more

readily take up DNA from their surrounding medium, and this DNA can be

stably integrated into the plant’s chromosomal genome

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EXPERIMENT NO. 8 - (PAPER - II):

Aim: Steps involved in the production of Bt cotton.

Principle: Major application of biotechnology is production of genetically modified

transgenic crops that are useful to mankind. Such crops are called GM crops in short.

These GM crops are mostly pest resistant, herbicide resistant and have more

nutritional value.

Bt cotton is a pest resistant GM crop. It is made to protect cotton crops from

ball worm. The Bt toxin producing gene introduced into cotton crop was taken from a

bacterium Bacillus thuringenesis.

Procedure (Steps involved in the production of Bt cotton):

1. The first step is identification of bollworm inhibiting gene, which was

identified in a bacterium called Bacillus thuringenesis.

2. Transgenesis (transfer of gene) is the next step. Agrobacterium mediated gene

transfer technique has been essentially used initially to make this GM crop.

However, direct gene transfer technique like biolistic gene transfer is being

used now.

3. Then, the GM plants are regenerated either from protoplasts or callus tissue.

4. The Cry gene, that produces crystal proteins (Cry proteins) was transferred to

cotton via Agrobacterium with CaMV35S promoter. The Cry protein of cotton

is an endotoxin and highly toxic to bollworms.

5. The GM cotton plants are developed from the callus tissue through somatic

embryogenesis.

6. Then the gene expression to the desired level is tested by growing the modified

crop in the field.

7. The important step used further is to produce new varieties by back cross

method.

8. Making Field trials is the final step to get approval for commercialization of the

new GM crop.

.……………………………….XXX…………………………………….

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EXPERIMENT NO.9 (PAPER - II):

Aim: Steps involved in the production of Golden Rice.

Principle: Golden rice is a variety of Oryza sativa, produced through genetic

engineering. The golden rice was created by introducing two β-carotene

biosynthesis genes into the plant. The golden rice is yellow in color and produces β-

carotene and give more quantity of vitamin-A.

Golden Rice technology is based on the simple principle that rice plants

possess the whole machinery to synthesise β-carotene, and while this machinery is

fully active in leaves, parts of it are turned off in the grain. By adding only two genes,

a plant phytoene synthase (psy) and a bacterial phytoene desaturase (CRT I), the

pathway is turned back on and β-carotene consequently accumulates in the grain.

Procedure (Steps involved in the production of Golden Rice):

1. The very first step in the golden rice development is identification of the genes

which can produce beta-carotene. These are phytoene synthase (psy from

daffodil) and a bacterial phytoene desaturase (CRT I from bacteria) and

lycopene betacyclase (Icy) from daffodil.

2. Transferring these genes into rice plant embryos is the next step. It is done by

Agrobacterium mediated transfer.

3. The rice embryos are precultured first and then they are grown along with

Agrobacterium.

4. Two vectors, one carrying psy and crtI genes, and the other carrying the gene

that code for Lycopene beta-cyclase, are used for co-transformation to

complete beta carotene biosynthesis.

5. The embryos incorporate the new genes into their DNA and produce desired

proteins. The transgenic embryos are grown in the tissue culture laboratory.

6. The plantlets are then transferred to green house for seed setting.

7. They are tested for the presence of beta-carotene after field growth. Fully

grown rice plants produced and stored beta-carotene in their starch.

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EXPERIMENT NO.10 (PAPER - II):

Aim: Steps involved in the production of transgenic plants producing edible vaccine.

Principle: Vaccines help in stimulating the antibodies production in human and

animals and provide immune protection against several diseases. Edible vaccines

refers to any foods; typically plants, that produce vitamins, proteins or other

nourishment that act as a vaccine against a certain disease. Edible vaccines are

genetically modified crops that contain added “immunity” for specific diseases.

Edible vaccines offer many benefits over traditional vaccines, due to their lower

manufacturing cost and a lack of negative side effects. Edible vaccines are currently

being developed for measles, Hepatitis B Hepatitis C, and cholera. Edible vaccines

are subunit vaccines; they contain the antigen proteins for a pathogen but lack the

genes for the full pathogen to form.

Procedure (Steps involved in the production of transgenic plants producing edible vaccine)

1. The first step in making an edible vaccine is the identification, isolation, and

characterization of a pathogenic antigen.

2. Once the antigen is identified and isolated, the gene is cloned into a transfer

vector. One of the most common transfer vectors for DNA being used for

edible vaccines is Agrobacterium tumefaciens.

3. The transgene can be expressed either through a stable transformation system

or through transient transformation system based on where the transgene has

been inserted into the cell.

4. A stable transformation involves a nuclear or plasmid integration in which

permanent changes occur in the recipient cells’ genes and the targeted

transgene is integrated into the genome of host plant cells.

5. Transient transformation involves a vector plasmid of Agrobacterium

tumefaciens which integrates the exogenous genes into the T-DNA, then infects

the vegetable tissue.

28

6. The vector is then turned into the desired strain with the help of the virulence

genes of the bacterium. It is then transferred and integrated into the genomic

DNA of the host plant by non-homologous recombination at random sites. It is

most effective for tomato, potato, and tobacco plants.

7. Another technique is the microprojectile bombardment method where selected

DNA sequences are processed and penetrated into the chloroplast genome. The

gene containing DNA coated metal particles are fired at the plant cells using a

gene gun. The plants take up the DNA, grow into new plants, then are cloned to

produce large numbers of genetically identical crops. The gene transfer is

independent, and it can express antigens through nuclear and chloroplast

transformation.

8. The pathogen sequence is inserted into the transfer DNA (T-DNA) to produce

the antigenic protein.

9. It is then inserted into the genome, expressed, and inherited in a mendelian

fashion. Then, the antigen is expressed in the fruit or plant.

10. Thereafter, traditional vegetative methods and techniques are used to grow the

plants and propagate the genetic line.

…………………………………….XXX………………………………….

29

EXPERIMENT NO.11 (PAPER - II):

Aim: Steps involved in the production of transgenic plants producing biodegradable

plastic.

Principle: scientists have recently genetically modified a plant, Arabidopsis thaliana,

so that it produces an organic flexible and moldable ‘plastic’ that is similar to

polypropylene, and can be used to make bags, bottles and containers.

Three new proteins were introduced into the plant which work with two proteins

which are normally present. Together these proteins make a material called

polyhydroxybutyrate-co-polyhydroxyvalerate (PHBV). The most useful site for

PHBV synthesis is chloroplast. The complete phb operon consists of 3 genes from

Ralstonia eutropha a bacterium. These are phbA, phbB, phbC.

Procedure: (Steps involved in the production of transgenic plants for producing

biodegradable plastic)

1. First step in making biodegradable plastic is to genetically modify the plant to

produce polyhydroxybutyrate (PHB) compound.

2. The genes - phbA, phbB, phbC that encode those enzymes which can

synthesize PHB (Polyhydroxybutyrate), are to be extracted from the bacterium

Ralstonia eutropha .

3. These genes are introduced into Arabidopsis thaliana plant through

Agrobacterium tumefaceins. These genes are easily expressed in Arabidopsis

plant.

4. The plastic like compound is formed by three enzymatic reactions. Glucose

coverts to acetyl CoA naturally in all plants. The first step is condensation of

two molecules of acetyl Co-A into acetoacetyl Co-A, which is catalyzed by β-

ketothiolase enzyme. This enzyme is coded by phbA gene.

5. The second enzyme acetyl Co-A reductase coded by the gene phb B, is

responsible for the conversion of acetoacetly Co.A into 3-Hydroxybutyryl

Co.A, by taking H from NADPH, which is oxidized to NADP.

6. Now, the enzyme phb synthase coded by phbC forms polyhydroxybutyrate by

polymerization reaction.

_

30

SPOTTING: (PAPER - I)

1. Autoclave:

1. Autoclave is an important instrument used to sterilize biological materials.

2. It is made up of steel and designed to withstand high temperatures and pressure.

3. The sterilization range of an autoclave is about 10 lb to 30 lb pressure and 120

to 275 0C temperature.

4. It ensures total destruction of microbial pathogens and makes the material fully

sterile.

5. It is normally used to sterilize culture media, cotton and cotton products, wool,

rubber gloves, plastic ware, metal tools with plastic handles and other materials

which cannot be sterilized in hot air oven.

2. Hot air oven:

31

1. The hot air oven is used for sterilizing microbiological material and other tools

used in biotechnology experiments.

2. It works by circulating very hot and dry air which destroys the microbes that

are present on the surface of materials.

3. The temperature ranges from 00C to 2000C.

4. It is used to sterilize glassware like petridishes, test tubes, beakers, pipette etc.

3. Laminar air flow:

1. Laminar air flow cabinet is a working station used to carry out tissue culture

experiments, where continuous and constant flow of air is present in one

dimension without any disturbance.

2. It is usually fitted with UV light and Prefilters like HEPA (High Efficiency

Particulate Air) Filters or ULPA (Ultra -Low Particulate Air) Filters.

3. The air flow velocity range from 0.3m/s to 0.5m/s towards the user.

4. It is used for compounding the sterile products in plant tissue culture.

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4. pH meter:

1. The pH of the medium plays very important role in the growth and

development of the organism and tissues.

2. The pH of the medium must be checked before incubating the explants in the

tissue culture experiment.

3. The pH is adjusted by adding either acid (HCl) or a base (NaOH).

4. pH meter is an electronic device to check the pH of liquid medium, consisting

of a column electrode, glass rod and a platinum wire. pH of the medium is

displayed on the digital screen.

5. Electronic balance

1. The electronic balance is normally used to weigh the chemicals that are used

to prepare nutrient medium.

2. It is a normal balance but with a digital display.

33

3. It gives quick and accurate weight, which is not possible in traditional

balances.

4. The precision of the balance relies on minute factors and wind, shaky

surfaces, or similar forces will cause the readings to be inaccurate.

6. Scalpel with blade / surgical scalpel

1. Scalpel is an essential tool used for making tissue dissections.

2. Scalpels may be single-use disposable or re-usable.

3. Re-usable scalpels can have permanently attached blades that can be

sharpened.

4. In tissue culture experiments the buds are cut from the shoot using scapel on

the working table of laminar air flow cabinet.

5. Surgical scalpels consist of two parts, a blade and a handle. The handles are

often reusable, with the blades being replaceable.Spatula

6. A spatula is a broad, flat, flexible blade used to mix, spread and lift any

material.

7. It is a stainless steel tool with two flat edges on a flexible blade.

8. It is used for transferring sub cultures, to keep chemicals in the balance.

9. They are resistant to deterioration from contact with boiling water, acids,

bases, and most solvents.

7. Scissors

1. Scissors are hand-operated tools that consist of a pair of metal blades with

handles. The blades pivot to bring the sharpened edges in contact with each

other, on action that cuts the material placed between the blades.

2. Scissors are used for cutting various thin materials, such as paper, metal or

aluminum foil, and wire.

3. It is used to cut roots, shoots from the aseptic Laboratory Spirit

4. Laboratory sprit or surgical spirit is used to clean the workstation surfaces,

and other tools used in the culture room.

5. It is also used to clean the user or workers hands.

6. It is used in the lamps called spirit lamp, which is useful in tissue culture

experiments.

7. The metal tools like forceps, scalpels, needles are cleaned with spirit and

heated on spirit lamp before use each time.

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8. Arrow headed sharp needle

1. Arrow headed sharp needle is another important tool used in tissue culture

laboratory.

2. It is used for dissecting out anthers from the flower.

9. Sodium hypochlorite

1. It is a chemical used as disinfectant.

2. It is used to prevent the tissue from infections.

3. It is a strong antiseptic that kills most forms of bacteria and viruses.

4. It is the most common chemical used for surface sterilization of explants.

10. Nutrient medium:

1. The basic requirement for growing any tissue is to provide nutrition

externally.

2. Different micro and macro nutrients are mixed in distilled water to make

liquid medium.

3. Vitamins and growth hormones like auxins and cytokinins are also added to

the medium to get plantlets.

4. Agar powder is added to make the medium solid.

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11. Agar:

1. Agar is most popular and good solidifying agent.

2. Agar is an inert material and cellular enzymes cannot digest it.

3. In tissue culture experiments the nutrient medium is made solid by using agar

powder.

4. It is obtained from some red algae members like Gelidium, Gracilaria .

12. Callus:

1. In tissue culture experiments callus is formed when basal nutrient medium is

used. i.e. nutrient medium without growth hormones.

2. The undifferentiated mass of tissue developed in culture is called "Callus".

3. Callus show organogenesis when plant growth hormones are added to the

medium.

4. To extract secondary metabolites, only callus culture is useful.

5. Callus is also useful to increase the number of tissue, which is later used in

sub-culture for micro-propagation

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SPOTTING: (PAPER - II)

1. Gene gun

1. A gene gun or biolistic particle delivery system is a device used to deliver

exogenous DNA (transgenes), RNA, or protein to cells.

2. The DNA is coated on either gold or tungsten particles.

3. These particles are loaded into the gun and they are accelerated to high speed

using electrostatic energy from pressurized helium or water droplet exposed

to high voltage.

4. The target tissue is usually callus or immature embryo growing on gel

medium.

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2. Micro pipette

1. Small sized pipettes that are capable of transferring very small amounts of

solutions.

2. Micropipette can transfer 0.2 microliters of the solution.

3. It is used under the microscope to inject liquid directly into the cell.

4. Micropipette is normally used in direct gene transfer into a single cell.

3. Electroporation

1. Electroporation is a technique used for direct DNA or gene transfer.

2. A foreign DNA is transferred or introduced into the plant cell by exposing

them for a very brief period of high voltage electric pulses.

3. These pulses induces transient (short-lived) pores in plasma lemma.

4. The external DNA (normally present in a solution) enters into the cell through

these pores (in the plasma membrane).

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4. Agrobacterium tumifaciens :

1. Agrobacterium tumifaciens is a gram negative, rod shaped, non-spore

producing soil bacterium, that has been extensively used for genetic

engineering. It causes gall / tumor formation at the base of the stem near the

ground.

2. It contains Ti (Tumor inducing) plasmid, which possess restriction enzymes

and is useful for the integration of foreign DNA. After integration it is called t-

DNA.

3. The selected gene is transferred into the bacterial plasmid, which then

integrates into the plant chromosome.

4. It is easy to insert any foreign DNA and integrated it to t-DNA.

5. Bt cotton

Bt cotton is a genetically modified organism (GMO) or genetically modified pest

resistant cotton variety, which produces an insecticide to combat bollworm.

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1. Bt stands for Bacillus thuringiensis

2. This bacterium produces Bt toxins that are insecticidal to the larvae of

bollworm.

3. The gene coding for Bt toxin has been inserted into cotton as a transgene,

causing it to produce this natural insecticide in its tissues.

6. Golden rice

1. Golden rice is a variety of rice (Oryza sativa) produced through genetic

engineering

2. The golden rice naturally biosynthesize beta-carotene, in the edible parts of

rice. Beta-carotene is a precursor of vitamin A.

3. Golden rice was created by transforming rice with two beta-carotene

biosynthesis genes viz. psy (phytoene synthase) from daffodil (Narcissus

pseudonarcissus) and crtI (phytoene desaturase) from the soil bacterium

Erwinia uredovora.

4. The psy and crtI genes were transferred into the rice nuclear genome and

placed under the control of an endosperm-specific promoter, so that they are

only expressed in the endosperm.

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7. Moon dust carnation

1. Moondust carnation, a GM (Genetically Modified) crop is a variety of

Dianthus species having purple mauve flower.

2. Its blue color comes from garden Petunia genes which are transferred into

the DNA of carnation.

3. Moondust is the first hybrid carnation created by Florigene, a biotechnology

company of Australia in the year 1994.

Acknowledgements:

The process of steps involved in the production of Bt Cotton was taken from the

monograph of CICR, Nagpur, (www.cicr.org.in) and http://isaaa.org.

The process of steps involved in the production of Golden Rice was taken from the

original paper by Peter Beyer et.al. The Journal of Nutrition, Volume 132, Issue 3,

March 2002, Pages 506S–510S

(the link is https://academic.oup.com/jn/article/132/3/506S/4687202)

41

Q 5. Identify and comment on the spot

Q 5. Identify and comment on the spot

42

Q 5. Identify and comment on the spot

Q 5. Identify and comment on the spot

43

Q 5. Identify and comment on the spot

Q 5. Identify and comment on the spot

44

Q 5. Identify and comment on the spot

Beaker

?

Q 5. Identify and comment on the spot

?

45

Q 5. Identify and comment on the spot

Q 5. Identify and comment on the spot

GONDWANA UNIVERSITY GADCHIROLI

BACHLOR OF SCIENCE(B.Sc.), SEMESTER – V

SUBJECT – ZOOLOGY

DISCIPLINE SPECIFIC ELECTIVES (DSE) CORE PAPER –XI

PAPER – III – INSECT VECTOR AND DISEASES

PRACTICAL MANUAL

BY

Dr Umesh Indurkar

Department Of Zoology,

Shri Dnyanesh Mahavidyalaya, Nawargaon

PRACTICAL MANUAL

1. Study of Life cycle of Mosquito, house fly, bed bug through ICT tools. Models or charts.

2. Study of different kinds of mouth parts of insects.

3. Study of following insect vectors through permanent slides/ photographs: Aedes, Culex,

Anopheles, Pediculus humanus capitis, Pediculus humanus, corporis, Phithirus pubis,

Xenopsylla cheopis, Cimex lectularius, Phlebotomus argentipes, Musca domestica.

4. Study of types of antennae through available permanent slides, charts or photographs.

5. Study of different diseases transmitted by above insect vectors.

6. Control appliances – sprayers and dusters.

7. Submission of a project report or case study on any one of the insect vectors and disease

transmitted.

Life cycle of Mosquito

The mosquito life cycle takes place in four distinct stages, from egg through to adult. Eggs Female mosquitoes lay eggs about every third day during their lifespan, usually in clumps of 100 to 300 eggs. The eggs are deposited either as “rafts” floating on the surface of standing water or on the ground in areas that regularly flood. Mosquitoes can lay eggs in as little as one inch of water. The eggs, generally white when laid, cannot hatch unless they are in water, usually for two to three days. Larvae When the eggs hatch, the larvae emerge. They are called “wigglers” because that’s how they swim. Most of the time, they hang from the surface of the water, breathing through tubes. The wigglers feed on organic matter in the water, shed their skins four times over about a week, and develop into pupae. Larvae are the easiest to kill, using oils that block their breathing or bacteria that poison them. Pupae The pupae are called “tumblers” for the way the fall into the deepest part of the water when threatened by predators. They are shaped like commas, partially encased in cocoons, with the head at one end and tiny flippers at the other. The pupae do not feed while developing, but breathe through tubes like the larvae. It takes about four days for the the adult mosquito to emerge.

Adults The newly emerged adults climb out of the water to rest and wait for their bodies to dry out. The males will take a day or two to fully develop their reproductive organs, then seek out a female, by the sound of her wingbeats, for mating. They’ll live about three to five days after that, feeding on fruit and plant nectar. The females mate once, but continue laying eggs after every blood meal. Under the best conditions, they can live up to a month or two.

Life cycle of house fly Musca Nebulo

The five stages involved in the life history of Musca Nebulo. The stages are: 1. Egg-Laying, 2. Egg 3. Larva, 4. Pupa.

1. Egg-Laying Four days after mating the female housefly lays her eggs. The housefly lays her eggs in stable manure by preference, but failing to find this, it may lay in human faeces, garbage or decomposing animal and vegetable matter. The conditions required for laying eggs are moisture and a favourable temperature, hence, stable manure or human faeces should not be dry. The female extends her ovipositor and lays about 120 to 160 eggs at one time. In the course of a breeding season a single female may lay eggs 4 to 6 times. 2. Egg An egg is whitish, cylindrical, and 1 mm long. It has two rib-like longitudinal thickenings on one side. The eggs hatch in 8 to 24 hours depending upon the temperature, and larvae emerge in the dung.

3. Larva The larvae are called maggots. They are highly modified without a distinct head, no thoracic or abdominal limbs, and with the spiracles greatly reduced in number. They are covered with thin soft chitin. Such a larva is known as an apodous larva.

The total larval period is from 6 to 8 days, during this time the larva moults twice, and it feeds and grows larger at each moulting. The larva in feeding moves away from light into moist and dark parts of the dung, it feeds on the substance in which it was hatched, it produces enzymes by which food is liquefied and it takes in liquids and small solid particles as food. 4. Pupa When the larva is ready to pupate it searches out a dry, dark crevice of the manure, the body contracts and segments are telescoped to form a pupa. Thus, the larva changes into a pupa without moulting, the last larval skin hardens to form an outer covering or puparium which encloses the pupa. Such a pupa is called coarctate. It has no chitinous covering of its own but only a soft pupal skin, the outer puparium has been formed by the last larval skin. The puparium is barrel-shaped and it becomes dark brown, externally it is segmented and shows traces of larval spiracles and spiny pads which become non-functional. The pupa takes in air by means of a pair of spine-like pupal spiracles projecting between the fifth and sixth segments of the puparium. The pupa is absolutely immobile, and the pupal stage lasts from 4 to 5 days. During this time internal changes take place, the larval organs are broken down or histolysis occurs by the phagocytes feeding upon the tissues of organs. The imaginal buds of the larva begin to form the organs of the adult, or histogenesis occurs in the pupa.

Life cycle of Bed bugs

Bed bugs are nocturnal, reddish-brown insects that feed on the blood of humans and other warm-blooded animals. These wingless insects have dorsoventrally flattened bodies that allow them to hide in areas such as floor cracks, carpets, beds and upholstered furniture. Eggs A bed bug's life begins with an egg, grain like and milky white in color. Female bed bugs lay between one and five eggs each day and may lie up to 500 eggs within one lifetime. Eggs are laid singly or in clusters and are placed within tight cracks or crevices. The egg is approximately 1 mm in length and is comparable in size to two grains of salt. Within two weeks, eggs hatch and immature bed bugs begin immediately to feed. Nymphs These young bed bugs, or nymphs, pass through five molts before reaching maturity. Although nymphs appear similar to adults, they are smaller in size and are not yet sexually mature. Young nymphs are also yellow-white in color, while older nymphs and adults are reddish-brown. In

order to complete a molting stage, each nymph requires a blood meal. At room temperature, nymphs molt and become adults within five weeks. Adults Upon reaching maturity, bed bug adults often make weekly feedings. When they’ve reached adulthood, bed bugs feed more frequently, usually about every 10 days. This is bad news for those of us who are unlucky enough to experience a bed bug infestation at home or on holidays. The adult life span is anywhere from 4-6 months to 18 months, so these little critters live for a very long life. Females can lay between 1 and 5 eggs each day – one of the reasons why these bugs can be so difficult to get rid of.

Insect mouthparts

• Labrum - a cover which may be loosely referred to as the upper lip. • Mandibles - hard, powerful cutting jaws. • Maxillae - 'pincers' which are less powerful than the mandibles. ... • Labium - the lower cover, often referred to as the lower lip. ... • Hypopharynx - a tongue-like structure in the floor of the mouth.

1. Biting and Chewing: This type of mouth parts are supposed to be the most primitive type as the other types are believed to be evolved from biting and chewing type of mouth parts. These consist of the labrum forming upper lip, mandibles, first maxillae, second maxillae forming lower lip, hypo pharynx and the epipharynx. The labrum is median, somewhat rectangular flap-like. The mandibles are paired and bear toothed edges at their inner surfaces; they work transversely by two sets of muscles to masticate the food. The first maxillae are paired and lie one on either side of the head capsule behind the mandibles. Each possesses a five-jointed maxillary palp which is a tactile organ.

The first maxillae help in holding the food. The second maxillae are paired but fused to form the lower lip. Its function is to push the masticated food into the mouth. The hypo pharynx is single median tongue-like process at whose base the common salivary duct opens. The epipharynx is a single small membranous piece lying under the labrum and bears taste buds.

This type of mouth parts are found in orthopteran insects like cockroaches, grasshoppers, crickets, etc. These are also found in silver fish, termites, earwigs, beetles, some hymenopterans and in caterpillars of Lepidoptera. 2. Chewing and Lapping: This type of mouth parts are modified for collecting the nectar and pollen from flowers and also for moulding the wax, as is found in honeybees, wasps, etc. They consist of the labrum, epipharynx, mandibles, first pair of maxillae and second pair of maxillae. The labrum lies below the clypeus, below the labrum is a fleshy epipharynx which is an organ of taste. Mandibles are short, smooth and spatulated, situated one on either side of the labrum; used in moulding wax and making the honeycomb. The labium (second pair of maxillae) has reduced paraglossae, the glossae are united and elongated to form the so called retractile tongue, at its tip is a small labellum or honey spoon. The labial palps are elongated. The glossa is used for gathering honey and it is an organ of touch and taste. The first pair of maxillae are placed at the sides of labium, they bear small maxillary palps, lacinia is very much reduced but galea are elongated and blade-like. The galea and labial palps form a tube enclosing the glossae which moves up and down to collect nectar from flower nectaries. The nectar is sucked up through the tube, so formed, by the pumping action of the pharynx. The labrum and mandibles help in chewing the food. 3. Piercing and Sucking: This type of mouth parts are adapted for piercing the tissues of animals and plants to suck blood and plant juice, and found in dipteran insects like mosquitoes and hemipteran insects like bugs, aphids, etc. They usually consist of labium, labrum and epipharynx, mandibles, maxillae (1st pair) and hypo pharynx. This type of mouth parts can be discussed in the following two headings: (i) Piercing and sucking mouth parts of mosquitoes: The labium is modified to form a long, straight, fleshy tube, called proboscis. It has a deep labial groove on its upper side. The labial palps are modified to form two conical lobes at the tip of the proboscis, called labella which bear tactile bristles. The labrum is long needle-like. The epipharynx is fused with the labrum. The labrum-epipharynx, thus, covers the labial groove dorsally from inside. These structures appear C- shaped in transverse section having a groove, called food channel. Mandibles, maxillae and hypo pharynx are modified to form needle-like stylets which are placed in the labial groove. In male mosquitoes, the mandibles are absent. The mandibles are finer than the maxillae, but both have saw-like edges on their tips. The hypo pharynx possesses salivary duct which opens at its tip. (ii) Piercing and sucking mouth parts of bugs: In bedbug, the labium constitutes a three- jointed proboscis. The mandibles and maxillae are modified to form stylets; the mandibular stylets possess blade-like tips, while maxillary stylets possess saw-like tips. The labrum is flap like and covers the labial groove at the base only.

Of the four stylets, mandibles are placed externally in the labial groove, while both the maxillae are placed internally in the labial groove. The maxillae are grooved and placed in such a way that they form an upper food channel and lower salivary canal. The epipharynx and hypo pharynx are absent. 4. Sponging: This type of mouth parts are adapted for sucking up liquid or semiliquid food and found in houseflies and some other flies. They consist of labrum- epipharynx, maxillae, labium and hypo pharynx; mandibles are entirely absent. In fact, in this type of mouth parts, the labium, i.e., lower lip is well developed and modified to form a long, fleshy and retractile proboscis. 5. Siphoning: This type of mouth parts are adapted wonderfully for sucking flower nectar and fruit juice, found in butterflies and moths belonging to the order Lepidoptera of class Insecta. They consist of small labrum, coiled proboscis, reduced mandibles and labium. The hypo pharynx and epipharynx are not found. The labrum is a triangular sclerite attached with the front clypeus of the head. The proboscis is formed by well-developed, greatly elongated and modified galeae of maxillae. It is grooved internally to form the food channel or canal through which food is drawn up to mouth. At rest, when proboscis is not in use, it is tightly coiled beneath the head but it becomes extended in response to food stimulus. The extension of proboscis is achieved by .exerting a fluid pressure by the blood. Mandibles are either absent or greatly reduced, situated on the lateral sides of the labrum. The labium is triangular plate-like bearing labial palps.

3. Study of following insect vectors through permanent slides/ photographs: Aedes, Culex, Anopheles, Pediculus humanus capitis, Pediculus humanus, corporis, Phithirus pubis, Xenopsylla cheopis, Cimex lectularius, Phlebotomus argentipes, Musca domestica

Aedes aegipty mosquito

Phylum: Arthropoda Class: Insecta Order: Diptera Family:Culicidae Genus:Aedes

Characters-Adult Aedes mosquitoes are distinguished from other types of mosquitoes by their narrow and typically black body, unique patterns of light and dark scales on the abdomen and thorax, and alternating light and dark bands on the legs. Females are further distinguished by the shape of the abdomen, which usually comes to a point at its tip, and by their maxillary palps (sensory structures associated with the mouthparts), which are shorter than the proboscis. Aedes mosquitoes characteristically hold their bodies low and parallel to the ground with the proboscis angled downward when landed.

Vector-Members of the genus Aedes are known vectors for numerous viral infections. The two most prominent species that transmit viruses are A. aegypti and A. albopictus, which transmit the viruses that cause dengue fever, yellow fever, West Nile fever, chikungunya, eastern equine encephalitis, and Zika virus, along with many other, less notable diseases. Infections with these viruses are typically accompanied by a fever, and in some cases, encephalitis, which can lead to death. A vaccine to provide protection from yellow fever exists, and measures to prevent mosquito bites include insecticides such as DDT, mosquito traps, insect repellents, and mosquito nets.

Culex Phylum: Arthropoda Class: Insecta Order: Diptera Family: Culicidae

Subfamily: Culicinae Genus: Culex Characters- Culex adults are usually drab, unicolorous mosquitoes, but some species of the subgenus Culex have markings on the legs and pale spots on the wings similar to Anopheles. Culex are characterised by the presence of distinct pulvilli and the absence of prespiracular setae and postspiracular setae. Culex larvae are distinguished from other genera by the following characters: seta 2-C usually absent; seta 3-C located on dorsal side of head, sometimes absent; palatal brushes normal, not developed for grasping prey; mandible normal, without lateral lobe at base; maxillary brush present, well developed; seta 12-I and comb always present; siphon with three or more pairs of prominent setae (seta 1-S); pecten normally present; saddle usually complete, sometimes incomplete and greatly reduced but never divided into dorsal and ventral sclerites or longer than the siphon; ventral brush (seta 4-X) usually with three or more pairs of setae. Vector-Diseases borne by one or more species of Culex mosquitoes vary in their dependence on the species of vector. Some are rarely and only incidentally transmitted by Culex species, • Arbovirus infections transmitted by various species of Culex include West Nile virus,

Japanese encephalitis and Western and Eastern equine encephalitis. and zika virus. • Nematode infections, mainly forms of filariasis may be borne by Culex species, as well as by

other mosquitoes and blood-sucking flies. • Culex mosquitoes can also transmit the parasitic disease lymphatic filariasis and the

bacterial disease tularemia.

Anophelus mosquitoe Phylum: Arthropoda Class: Insecta

Order: Diptera Family: Culicidae Subfamily: Anophelinae Genus: Anopheles Characters-Anopheles mosquitoes can be distinguished from other mosquitoes by the palps, which are as long as the proboscis, and by the presence of discrete blocks of black and white scales on the wings. Adults can also be identified by their typical resting position: males and females rest with their abdomens sticking up in the air rather than parallel to the surface on which they are resting The head is specialized for acquiring sensory information and for feeding. It contains the eyes and a pair of long, many-segmented antennae. The antennae are important for detecting host odors, as well as odors of breeding sites where females lay eggs. The head also has an elongated, forward-projecting proboscis used for feeding, and two maxillary palps. These palps also carry the receptors for carbon dioxide, a major attractant for the location of the mosquito's host. The thorax is specialized for locomotion. Three pairs of legs and a pair of wings are attached to the thorax. The abdomen is specialized for food digestion and egg development. This segmented body part expands considerably when a female takes a blood meal. The blood is digested over time, serving as a source of protein for the production of eggs, which gradually fill the abdomen. Vector- Members of the protozoal species Plasmodium, which cause malaria, have evolved a successful relationship with their arthropod vector, the Anopheles mosquito also transmitted the diseases Lymphatic filariasis

.

Pediculus humunus

Phylum: Arthropoda Class: Insecta Order: Phthiraptera Family: Anoplura Genus: Pediculus Species: P. humanus Characters- Adult head lice are small (2.5–3 mm long), dorsoventrally flattened and entirely wingless. The thoracic segments are fused, but otherwise distinct from the head and abdomen, the latter being composed of seven visible segments. Head lice are grey in general, but their precise color varies according to the environment in which they were raised. After feeding, consumed blood causes the louse body to take on a reddish color. Head One pair of antennae, each with five segments, protrudes from the insect's head. Head lice also have one pair of eyes. Eyes are present in all species within the Pediculidae (the family of which the head louse is a member), but are reduced or absent in most other members of the Anoplura suborder. Thorax Six legs project from the fused segments of the thorax. legs are short and terminate with a single claw and opposing "thumb". Between its claw and thumb, the louse grasps the hair of its host. With their short legs and large claws, lice are well adapted to clinging to the hair of their host. Abdomen Seven segments of the louse abdomen are visible. The first six segments each have a pair of spiracles through which the insect breathes. The last segment contains the anus and the genitalia Vector-The human body louse, Pediculus humanus corporis is the primary vector of the bacterial agents of louse-borne relapsing fever, trench fever, and epidemic typhus. Epidemic typhus, one of the most significant historical diseases of humans, is caused by Rickettsia prowazekii. Besides its notoriety as the agent of the recurrent chronic disease, trench fever, Bartonella quintana can cause endocarditis and is a common infection among the homeless. Borrelia recurrentis causes another recurrent fever in central and Eastern Africa that is characterized by significant morbidity and mortality.

Xenopsylla cheopis flea Phylum: Arthropoda Class: Insecta Order: Siphonaptera Family: Pulicidae Genus: Xenopsylla Species: X. cheopis Characters-The flea's body is about one tenth of an inch long (about 2.5 mm). Its body is constructed to make it easier to jump long distances. The flea's body consists of three regions: head, thorax, and abdomen. The head and the thorax have rows of bristles (called combs), and the abdomen consists of eight visible segments. They are small insects, 1–5 mm; the body is flattened laterally, generally light brown to dark brown, with modified posterior legs for jumping, no wings and reduced antennae. A flea's mouth has two functions: one for squirting saliva or partly digested blood into the bite, and one for sucking up blood from the host. This process mechanically transmits pathogens that may cause diseases it might carry. Fleas smell exhaled carbon dioxide from humans and animals and jump rapidly to the source to feed on the newly found host. The flea is wingless so it cannot fly, but it can jump long distances with the help of small, powerful legs. A flea's leg consists of four parts: the part that is closest to the body is the coxa; next are the femur, tibia, and tarsus. A flea can use its legs to jump up to 200 times its own body length (about 20 in or 50 cm). Vector- Fleas are vectors for viral, bacterial and rickettsial diseases of humans and other animals, as well as of protozoan and helminth parasite. Bacterial diseases carried by fleas

include murine or endemic typhus and bubonic plague. Fleas can transmit Rickettsia typhi, Rickettsia felis, Bartonella henselae and the myxomatosis virus. They can carry Hymenolepiasis tapeworms and Trypanosome protozoans. The chigoe flea or jigger (Tunga penetrans) causes the disease tungiasis, a major public health problem around the world. Fleas that specialize as parasites on specific mammals may use other mammals as hosts; thus, humans may be bitten by cat and dog fleas.

Cimex(Bed bugs)

Characters-Adult Cimex are light brown to reddish-brown, flat, oval, and have no hind wings. The front wings are vestigial and reduced to pad-like structures. Adults grow to 4–5 mm long and 1.5–3 mm wide Bed bugs have five immature nymph life stages and a final sexually mature adult stage They shed their skins through ecdysis at each stage, discarding their outer exoskeleton. Newly hatched nymphs are translucent, lighter in color, and become browner as they moult and reach maturity. Vector-Symptoms may not appear until some days after the bites have occurred. Reactions often become more brisk after multiple bites due to possible sensitization to the salivary proteins of the bed bug. The skin reaction usually occurs in the area of the bite which is most commonly the arms, shoulders and legs as they are more frequently exposed at night. Numerous bites may lead to an erythematous rash or urticaria. It is also possible that sustained feeding by bedbugs may lead to anaemia. It is also important to watch for and treat any secondary bacterial infection

Plebotomus Sand fly Characters- Sandfly is about 8.5mm in length has a brownish colour during the day, it has hairs that are close to each other that coat it. The wings are “V” shaped. The two wings have dense hairs and increases pigmentation patterns. The large compound eyes are more or less contiguous above the bases of the 15-segmented antennae. The pedicel of the males' antennae houses the Johnston's organ. The mouthparts are well-developed with cutting teeth on elongated mandibles in the proboscis, adapted for blood-sucking in females, but not in males. The thorax extends slightly over the head, and the abdomen is nine-segmented and tapered at the end. Disease Transmitted

1. Visceral leishmaniasis also known as kala-azar, black feve and Dumdum fever

2. Cutaneous leishmaniasis also known as oriental sore, tropical sore, chiclero ulcer, chiclero's ulcer, Aleppo boil, Delhi Boil or desert boil)

3. Phlebotomus fever (also known as Pappataci fever an, sandfly fever and three-day fever)

Musca domestica Phylum: Arthropoda Class: Insecta Order: Diptera Section: Schizophora Family: Muscidae Genus: Musca Species: M. domestica Characters-Adult houseflies are usually 6 to 7 mm long with a wingspan of 13 to 15 mm. The females tend to be larger winged than males, while males have relatively longer legs. Females tend to vary more in size and there is geographic variation with larger individuals in higher latitudes. The head is strongly convex in front and flat and slightly conical behind. The pair of large compound eyes almost touch in the male, but are more widely separated in the female. They have three simple eyes (ocelli) and a pair of short antennae. Flies land on a ceiling by flying straight towards it; just before landing, they make a half roll and point all six legs at the surface, absorbing the shock with the front legs and sticking a moment later with the other four The thorax is a shade of gray, sometimes even black, with four dark, longitudinal bands of even width on the dorsal surface. The whole body is covered with short hairs. Like other Diptera, houseflies have only one pair of wings; what would be the hind pair is reduced to small halteres that aid in flight stability. The wings are translucent with a yellowish tinge at their base Each wing has a lobe at the back, the calypter, covering the haltere. The abdomen is gray or yellowish with a dark stripe and irregular dark markings at the side. It has 10 segments which bear spiracles for respiration. In males, the ninth segment bears a pair of claspers for copulation, and the 10th bears anal cerci in both sexes. Vector-Houseflies can fly for several miles from their breeding places, carrying a wide variety of organisms on their hairs, mouthparts, vomitus, and faeces. Parasites carried include cysts of protozoa, e.g. Entamoeba histolytica and Giardia lamblia and eggs of helminths, e.g., Ascaris lumbricoides, Trichuris trichiura, Hymenolepis nana, and Enterobius vermicularis. Houseflies do not serve as a secondary host or act as a reservoir of any bacteria of medical or veterinary importance, but they do serve as mechanical vectors to over 100 pathogens, such as those causing typhoid, cholera, salmonellosis, bacillary dysentery, tuberculosis, anthrax, ophthalmia,

and pyogenic cocci, making them especially problematic in hospitals and during outbreaks of certain diseases. Disease-causing organisms on the outer surface of the fly may survive for a few hours, but those in the crop or gut can be viable for several days. Usually, too few bacteria are on the external surface of the flies (except perhaps for Shigella) to cause infection, so the main routes to human infection are through the fly's regurgitation and defecation

4. Study of types of antennae through available permanent slides, charts or photographs.

Types of insect antennae

Aristate Capitate Clavate Filiform Flabellate

Geniculate Setaceous Lamellate Moniliform Pectinate

Plumose Serrate Stylate

The Types of Antennae – The antennae are highly modified in different groups of insects. They

possess the taxonomic and sexual dimorphic significance in some insects, particularly the Coleoptera and Diptera.

They can be classified into 13 types as follows: (i). The Setaceous Antennae. The flagellum looks like a bristle due to the gradual reduction in the size of the segments, e.g., Odonata.

(ii) The Filiform ntennae. The flagellum appears as a threadlike structure made up of uniformly thin segments, e.g., Blattidae. (iii) The Moniliform Antennae. The flagellum is composed of the globose-shaped segments, providing a beaded or necklace-like appearance to the entire antennae, e.g., Termites, Calotermes. (iv) The Serrate Antennae. The flagellum consists of triangular segments with eccentric arrangement leaving the free ends of the segments freely on one side which appear as the teeth of a saw, e.g., Buprestidae, Elateridae (Coleoptera). (v) The Pectinate or Flabellate Antennae. The individual segments of the flagellum are extending on only one side of the long processes and thus antennae become comb-like. e.g., Rhipiphoridae (Coleoptera), Tenthredinidae (Hymenoptera), Bombycoidea (Lepidoptera). (vi). The Clavate Antennae. The successive segments of the flagellum become qradually broader giving an appearance of the club-shaped form to the antennae, e.g. Siphidae (Coleoptera), Lepidoptera. (vii). The Capitate Antennae. The proximal segments of the flagellum are of uniform size while the distal segments modify into a large knob or capitulum, e.g. Nniulidae (Coleoptera). (viii). The Lamellate Antennae. The terminal segments flagellum modify into the leaf- like broad plates forming a foliate capitulum, e.g. Melolontha, Scarabaeidae (Coleoptera). (ix) The Geniculate Antennae. The antennae are bent completely from the scape-pedic joint, e.g., Chalcidoidea. (x) The Plumose of Antennae. The flagelum is composedof large number of cylindrical segments and the segments are provided with long hairs on either side, e.g., male mosquitoes. (xi) The Pilose Antennae. The flagellum looks like a shaft, flexible hair., All segments are alike, fine and tubular but without processes. e. g. female mosquitoes. (xii) The Aristate Antenna. Tie flagellum is undivided but the second segment is situated at the different angle on the first segment and the antenna becomes pendant rather than usual. The arista develops on the third segment, i.e., first segment of the flagellum. The arista may be bare or hairy. The hairs may be developed on either side of arista as seen in house fly . Whenever the hairs are developed above the arista, in the tsetse fly. (xiii). The Stylate Antennae. Three segmented antennae and third segment divided repeatedly and thus it become falsely multi segment often farming a stylate. Brachera Tabanus.

1. Control appliances – sprayers and dusters

Uses of spraying and dusting equipments • For the insecticides application to control insect pests on crops and in stores, houses, kitchens,

poultry farms, barns, etc. • For the acarices application to control phytophagous mites. • For the fungicides and bactericides application to control the plant diseases. • For the herbicides application, to kill the weeds

• For the harmone sprays application to increase the fruit set or to prevent the premature dropping of fruits.

• For the application of plant nutrients as foliar spray. • For applying the powdery formulation of poisonous chemicals on the crops and houses, kitchens.

Types of dusters • Depending on the source of power it can be classified as manually operated and power operated

dusters. • The manually operated dusters are (i) package duster (ii) plunger duster (iii) bellow duster and

(iv) rotary duster. (i) Package dusters

• In some pesticide dusts are packed in containers that serve as a hand applicators and may be discard after use.

• They are mostly provided with rubber, leather or plastic section which, on getting squeezed, provides a puff of air that emits the dust in a small cloud.

• The simplest type of package duster is worked by pressing it between the fingers. (ii) Plunger dusters

• The consists of an air pump of the simple plunger type, a dust chamber, and a discharge assembly consisting of a straight tube or a small exit pipe whose discharge outlet can be increased or decreased by moving a lid provided at the end of the dust chamber.

• The air from the pump is directed through a tube into the container where it agitates the dust and eject it from a discharge orifice or tube.

• The amount of dust can be controlled by the speed of the operation of the pump. • These are useful for spot application in restricted areas and for controlling ants, poultry pest and

pest of farm animals. (iii) Rotary duster

• A consists basically of a blower complete with a gear box and a hopper. It is operated by rotating the crank.

• The cranking motion is transmitted through the gear box to the blower. • A drive is taken for the dust agitator located in the hopper. • The rotary duster may be hand carried type or shoulder mounted or belly carried type. • The feed is controlled by a feed control lever, which operates a slide to control the aperture at the

bottom of the hopper. (iv) Power dusters

• The resemble the rotary duster is construction, except that the power to drive the blower through

the gear box is tapped from an external power source which may be an engine or P.T.O. shaft of the tractor or flywheel of the power tiller.

• The power operated centrifugal energy knapsack sprayer also can be converted into a power duster, by allowing the dust fluid into the air stream, near the point of attaching the pleated hose, in the blower elbow. Sprayers

• The Sprayer is one which separates the spray fluid (which may be a suspension, an emulsion or a solution) into small droplets and ejects it with little force for distributing it properly.

• It also regulates the amount of pesticide to avoid excessive application that might prove wasteful or harmful.

• The mechanical appliances that are used for distributing the dust formulations of pesticides are called as dusters. Hand sprayer

• The hand sprayer is a small, light and compact unit. • The capacity of the container varies from 500 to 1000 ml. • This is generally used for spraying small areas like kitchen garden and experimental laboratory

plots. • It is a hydraulic energy sprayer. • It has a hydraulic pump inside the container, with cylinder, plunger and a plunger rod. • By operating the plunger up, the spray fluid in the container is sucked into the cylinder through a

ball valve assembly and then pressurised during the downward stroke. • The pressurised fluid is then let out through a nozzle, and sprayed into fine droplets. • If the pressure to be built inside the container an air pump with cylinder, plunge and plunger rod

is required. • When the plunger is pulled up, the air is sucked into the cylinder and when pushed down the air

bubble is releases into the container with 80% of its volume filled with the fluid. • The air reaches the space above the free fluid surface and presses the fluid. • The pressurised fluid is drawn up through a trigger cut of valve to the nozzle, where is atomized

and sprayed. • In some other type, air pump and the container are separate pieces and the pump is attached to

the container is such a way to release the pressurised air through an orifice at the top of the container.

• The fluid is lifted through an office at the top of the container. • The fluid is lifted through a capillary tube due to surface tension developed by the high velocity

air at the outlet and sheared away by the air and sprayed as droplets. Foot sprayer

• This is a modified version of rocker sprayer.• The pump is fixed in a vertical position with necessary braces.• The plunger moves up and down when operated by the pedal.• A ball valve is provided in the plunger assembly itself to allow the fluid to cross the plunger and

getting pressurized in the pressure vessel.• During the upward motion of the piston fluid is sucked in and pressurized into the pressure

vessel and during downward movement, the sucked fluid crosses the plungers and enter thepump.

• The pressure developed is about 17-21 kg/cm2.Bucket sprayer

• The bucket sprayer is designed to pump the spray fluid directly from, the open container, usuallya bucket.

• The hydraulic pump will be put inside the bucked and held properly with the help of foot rest.• As the plunger is pulled up, the fluid enters through the suction ball valve assembly and when the

plunger is pressed down, the suction valve closes and the fluid enters the pressure chamberthrough a ball valve assembly.

• As the plunger is continuously worked, pressure is built in the pressure chamber and the deliveryhose.

• As soon as the required pressure is built up, the spraying will be done.• A pressure of 4 kg / cm2 is developed in most of the models.