A DESCRIPTIVE REPORT ON THE INDUSTRIAL AFFAIRS AT THE KENYA AGRICULTURAL AND

LIVESTOCK RESEARCH ORGANIZATION (KALRO) NAKURU IN THE DEPARTMENT OF AGRICULTURE

AND LIVESTOCK.A PERIODICAL ACCOUNT FROM 05.08.2023 TO 06.30.2023 SUBMITTED BY
RARYEA MOSES AS PARTIAL COMPLETION OF THE REQUIREMENTS FOR THE GRADUATION OF A
BSC IN MICROBIOLOGY.

SCHOOL: BIOLOGICAL SCIENCES

DEPARTMENT: BOTANY

NAME: RARYEA MOSES

REG NO: SCB212-0213/2020

SUPERVISOR: Mr. M. Wambura

DATE OF SUBMISSION: 30THJULY 202

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DECLARATION

This attachment report was written for the training I undertook at KALRO-Nakuru
County. It is entirely original to me and has never been presented in any other setting.

AFFIRMATION

NAME: …………………………………. SIGN: …...…………………

DATE: …………………………………...

UNIVERSITY BASED SUPERVISIOR

NAME: ………………................... SIGN: ………………………….

DATE: …………………………………...

INDUSTRY BASED SUPERVISIOR

NAME: …………………… SIGN: ………………………….

DATE: …………………………………...

ACKNOWLEDGEMENT

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I had a great experience working at KALRO from May to June as an attaché. My work

as a microbiologist and biotechnologist will undoubtedly be shaped and influenced by

the fun and experiences I've had. I have emphasized in this report the fantastic

potential that KALRO provides to young scientists from various colleges around the

nation. The entire KALRO family's contributions and cooperation were essential to the

production of this study. My sincere gratitude is extended to the Almighty God, who

gave me the physical and mental stability I needed to finish my attachment, as well

as to the research team in the biotechnology lab, which is led by Cyrus Kimani

Ndungu, as well as to Madams Beatrice and Wandia from the cereal chemistry lab,

Madam Lorna Chesir from the soil chemistry lab, and Dr. Zenah, head of the plant

pathology lab. God bless you all for sharing your invaluable expertise in experiments

and data analysis.

LIST OF ABBREVIATIONS AND ACRONYMS

ABBR 1: KALRO- KENYA AGRICULTURAL AND LIVESTOCK RESEARCH ORGANIZATION

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DEFINITION OF TERMS

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EXECUTIVE SUMMARY

This particular attachment report provides a thorough assessment, analysis, and

interpretation of the numerous laboratory diagnostic techniques used by the Kenya

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Agricultural Livestock and Research Organization. It describes all the tests carried out

in detail and outlines the fundamental concepts of diagnostics, nucleic acid

extraction, and culture techniques, all of which are crucial in scientific research

projects. The laboratories were used in a rotational fashion for a period of two weeks,

and the standard goals were to: ease the transition from school to the workplace;

acquire practical skills and support application of theoretic knowledge; comprehend

the role of research in communicating scientifically based findings towards problems

affecting the community; and comprehend the role of biotechnology in research. Also,

to build mutually beneficial relationships with other scientists at various research

institutes and to share expertise, knowledge, and diverse biotechnology technologies

SECTION ONE: INTRODUCTION- ORGANIZATIONAL PROFILE

GEOGRAPHICAL LOCATION

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 The Food Crops Research Centre Njoro of the Kenya Agricultural and Livestock

Research Organization (KALRO) is situated 20 kilometers south-west of Nakuru in the

Western Rift Valley. It is located 200 kilometers from Nairobi in the Njoro Sub-County

of Nakuru County at a height of 2120 meters above sea level.

HISTORICAL BACKGROUND

The Act of 2013 established the Kenya Agricultural and Livestock Research Organization as

a corporate body with the goal of creating an appropriate institutional and legal framework

for conducting extensive agricultural and livestock research. The Act gave the cabinet

secretary the authority to create any research institutes that may be required for KALRO to

carry out its mandate under the Act, after consulting with the KALRO Board. The

organization, which now oversees 18 research institutes across the nation, combined with

KARI, Coffee Research Foundation, Tea Research Foundation, and Kenya Sugar Research

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Foundation. The objectives of KALRO are to facilitate equitable access to research

information, resources, and technology while also promoting the use of research findings

and technology in the agricultural sector. These objectives also include promoting,

streamlining, coordinating, and regulating research in Kenya pertaining to crops, livestock,

genetic resources, and biotechnology.

The Act of 2013 allowed for the establishment of four KALRO centers, which became

operational on July 1st, 2014. These facilities are KALRO Kandara (Muranga), KALRO Matuga

(Kwale), KALRO Kibos (Kisumu), and KALRO Tigoni (Kiambu).

VISION

Excellence in agricultural and livestock research towards transformed livelihood

MISSION

To conduct agricultural research through application of science, technology and

innovation to catalyze sustainable growth and development in agriculture and livestock

Product Value Chains.

CORE VALUES

1. Customer orientation: the central focus of KALRO is to provide timely and

responsive demand-driven research interventions aimed at addressing the needs

of the customers within the agricultural sector. KALRO will achieve this by
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 maintaining a culture that promotes responsiveness to customer needs.

2. Professionalism: KALRO will uphold high standards of excellence in the

provision of services to their customers. One of the hallmarks of excellence is the

40 Kenya Agricultural & Livestock Research Organization integrity and ethics in all

areas of operation. In this regard, KALRO’s research outputs and

recommendations will adhere to the highest standards of scientific rigor, ethics,

and sound evidence base.

2. Innovativeness: KALRO recognizes the need for innovation in order to be responsive to

the agricultural sector challenges. In this regard, KALRO commits

to develop flexible and integrated solutions in tandem with the needs of customers and

stakeholders.

4. Collaboration: Given the multi-stakeholder interest that the agricultural sector

attracts, KALRO is expected to collaborate and forge partnerships for the

development of the sector in Kenya and beyond. KALRO will therefore endeavor to create

beneficial opportunities for agricultural research and development.

5. Environmental consciousness: KALRO will ensure that the environment is conserved

while discharging its mandate.

6. Integrity: KALRO is committed to ethical delivery of its mandate to all stakeholders.

ORGANIZATIONAL STRUCTURE

A Board of Directors oversees KALRO's operations. In addition to two (2) Deputy Director

Generals and sixteen (16) Institute Directors, the Director General serves as the Chief Executive
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Officer (CEO). KALRO has sixteen institutes. 51 centers are thoughtfully dispersed across the

nation to accommodate various academic fields, agro-ecological regions, and socioeconomic

systems.

DETAILS OF PLACEMENT SECTION(S)/ DEPARTMENT(S)

Exposing students to biotechnology and biosafety applications in research and industry is the

goal of the industrial attachment. Students will complete an eight-week supervised internship in

a variety of work settings pertaining to biotechnology, food safety, plant pathology, or other

related fields. They will have to take part in the institution's technological operations and

activities. Technical reports and logbooks detailing their station activities will be required of

them. Their daily activity logbooks will be turned in for evaluation.

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 SECTION TWO: ATTACHMENT EXPERIENCES

CEREAL CHEMISRTY LABORATORY

The Kenya Agriculture Livestock Research Organization's (KALRO) Food Quality Control

Laboratory, run by the ministry of agriculture, is responsible for identifying the nutritional

and biochemical qualities of fresh and processed food samples. The facility is utilized by

researchers, business owners, students, and the food industry. Operators from each of

Nakuru's districts. According to the Kenya Bureau of Standards (KEBs), nutrient labeling is a

necessary component of value-added and processed products. Research on nutrient content

in various food samples, such as levels of cyanide, phytic acid, vitamins, and proteins in

various types of food samples and cereals, is carried out by the Food Quality Laboratory

(FQL).

1 Instrumentation
Food Testing To assess the integrity and caliber of solid and liquid food samples, laboratories need
specialized equipment. Consumers are highly concerned about food safety, and testing in the lab
or on the field is the first step toward ensuring it in the future. Food testing is essential for
ensuring food safety, but it is also useful for routine quality control. A table outlining the many
types of laboratory equipment can be seen below:

vortex machine This facilitates sample mixing more

quickly.

Spectrophotometer used to calculate the moisture content

of flour and wheat grain.
boerner divider A Boerner-type divider is a device that
divides a grain sample into two smaller,
equal parts using gravity. The valve in
the hopper throat is opened to release
the sample once it has been placed in
the top hopper.
farinograph used to calculate the amount of
moisture in the dough

Fermentation unit used to speed up the dough's

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 fermentation process
Kjedahl used to calculate the overall protein
content of food samples.
Dough mixer Using this, the proper amount of flour,
water, and other ingredients are added
to the dough and blended.

Hectoliter used to calculate the sample's density

Oven For baking bread

Food Nutrient Analysis

Finding out a food's nutritional value is done through food nutritional analysis. It is an essential

component of the cereal chemistry lab that offers details on the chemical make-up, processing,

quality assurance, and contamination of foods including wheat, maize, and whole grains.

Picrate Test for Cyanide test in cassava

In cassava, cyanide is linked to a glucoside in the form of 93% limarin and 7% lotaustralin.

From the peel to the parenchyma and from the distal to the proximal end of the tuber, the

concentration of cyanide decreases.

As plants age, levels start to decline and are highest in young leaves and petioles.

The picrate leaf test is easy to perform, takes only a few minutes, and only a few low-cost

supplies are needed. Depending on labor availability, the technique enables the

examination of hundreds of plants per day. The main benefit of the test is that it makes it

possible to screen cassava seedlings before the roots become huge.

(i) Using a rubber stopper as a base cot 2-cm leaf disc with a cork

borer, choose young but fully expanded leaves. Test one leaf each

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from at least four plants per clone.

(ii) With forceps, place leaf disc into a glass vial (25 mm outer diameter,

84 mm high).

(iii) Pipette a drop of tuolene onto the leave disc.

(iv) Dip filter paper (0.7cm x 1.0cm) into sodium picrate solutionwhich is

prepared by mixing equal volumes of 5% sodium carbonate solution

and saturated solution of picric acid. Suspend the filter paper from

the cap of the vial without touching ihe leaf disc. The filter paper will

gradually change from yellow to red, the shade depending on the

amount of hydrogen cyanide released from the leaf disc.

(v) Let the set up stand for vor hours, then note the colour's intensity. The more intense

the colour, the more cyanide the leaf contains. Selection for low cyanide clones should be

based on the average rating of at least four plarnts per clone

METHODOLOGY FOR RAPID EVALUATION OF CYANIDE CONTENT OF CASSAVA ROOTS.

1. Prepare alkal1ne picrate solution sa follows. Dissolve 25g anhydrous Na,co, and 5g moist

pictrate acid in distilled H,o (essentially a saturated solution; may be maintained for

several months).

2. Cut lx 6 cm strips of whatman No I qualitative filter paper.

3. Dip filter paper strip into stock picrate and drain free from excess liquid just before use.

4. Place Igm sample of cassava, root in test tube.

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5. Add 5 drops of toluene.

6. Immediately place filter paper saturated with alkaline pictrate suspended above sampie

by holding in place with plastic cap. (avoid contact between strip and sample side tube.)

7. Leave at room temperature for 24 hrs.

8. Rate on a l-5 or |-9 on the basis of intensity of red colour (brighter intensity of red

higher HCN content of root sample.)

Approximation of root HCN content:

Rating HCN Content (ppm)

1 =< 10

2 =10-15

3 =15-25

4 -25-40

5 =40-60

6 =60-85

7 =85-115

8 =115-150

9 >150

New score structure

1-4 = 0-50

5-6 = 50-100

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7-9 >100

ROOTS

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The sample on the first image changed color from blue to red-brown due to the reaction

between toluene and hydrogen cyanide. Cyanide ions were barely detectable in the

samples on the right, if at all.

Cassava leaf

10 range ppm- m97

20 range ppm- karembo

Cassava roots
5 range ppm- m97
200 range ppm-karembo

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PHYTIC ACID TEST
Phytic acid is a well-known naturally occurring compound that can be found in plant seeds, edible seeds, and
legumes. Phosphorus is stored in grains like maize, beans, green grams, etc. as phytic acid. Phytate, which is
phytic acid attached to a mineral in a seed, is broken down during seed germination and phosphorous is
released to be used as an energy source. The following procedure was followed when phytic acid was
extracted:

•1.0g of dried soya bean sample was weighed

•1.5ml was transferred into microfuge tubes.
•0.4M of HCL was added and incubated for 24 hours in order to extract phytic acid present in the sample.
•The sample was vortexed and 20 μl of the sample was transferred into microliter plate.
•The sample was supplemented with 180 microliter of Chen’s reagent and mixed thoroughly.
•The results were observed and recorded

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Calculation of Concentrations

If 0.174grams of KzHPO4 : are dissolved in I litre of water i.e. stock solution of

0.174grams in 1,000,000 ul. of Water.

Then how much of the salt is contained for example in I5 l.

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=),174*15 μL / 1000000 μL to give

0.0000026'g of K2HPO4 =15 μL

2.61 »g in 15 μL

Kjedahl protocol for Nitrogen and protein analysis

When soil is treated with hydrogen peroxide, sulfuric acid, selenium, and boric acid, a digest is
produced. This digest is used to assess the total nitrogen concentration. By combining nitrates
with boric acid in an acid medium to generate 4-nitrosalicyclic and/or 3-nitrosalicyclic, the main
concept accounts for the potential omitting of nitrates. The organic matter in the soil reduces the
compounds to the proper amino acid forms, and complete oxidation of the organic matter is
necessary for the determination of total nutrients. The organic matter is first oxidized by hydrogen peroxide,
which is then catalyzed by a selenium compound. At high temperatures, sulfuric acid then completes the digestion. The
primary benefits of this approach are that practically all nutrients must be dissolved in solution after a single digestion;
there is no metal or nitrogen volatilization; and the approach is straightforward and quick. For the analysis of soil
nitrogen, the following procedure was followed:

Reagents:
 Se powder (selenium).
 Digestion mixture, commonly known as a mixture of sulfuric acid and selenium powder.
 To make this mixture, weigh 2.8g of selenium powder, add 800ml of concentrated sulfuric
acid, then heat the mixture until the selenium suspension changes from its initial blackish
hue to a clear light yellow after passing through a green blue phase.
 Sodium Hydroxide, 3.4%. Dissolve 400g of sodium hydroxide pellets in 1000ml of distilled
water to make this.
 4.2% Boric Acid. Dissolve 20g of boric acid in 1000ml DW.
 5.0.01N Hydrochloric acid dilute 8.6ml of concentrated Hydrochloric acid in 100ml, then
dilute 100ml of that to 1000ml mark with DW.

Protocol:
 The soil was weighed at 1.0g, 4ml of the digestion mixture was added, and the mixture was
digested at 110.0°C for an hour and then at 330.0°C for two hours. The remedy seemed to
be colorless.
 The items were given time to cool.
 Distillation was carried out while collecting the distillate in a receiver, a 50ml conical flask
containing 25.0ml boric acid with drops of mixed indicator. • 25.0ml of distilled water was
added, followed by 25.0ml of sodium hydroxide.
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  0.01N HCL was used for back titration.
 Free ammonia was extracted from the solution by steam distillation and titration in the
presence of too much sodium hydroxide. The distillate was collected in a receiver conical
flask with drops of mixed indicator and excess boric acid.
 Distillation and titration of free ammonia was liberated from solution by steam

distillation in presence of excess Sodium hydroxide. The distillate was collected in a

receiver conical flask containing excess boric acid with drops of mixed indicator.

Blank 0.6
119- 12.5
120- 10.0
121- 38.5

((12.5-0.6)* 0.01 * 14.007/ 1.000* 1000) *100

= 0.166%

((10.0-0.6)* 0.01 * 14.007/ 1.000* 1000) *100

= 0.1317%

((38.8-0.6)* 0.01 * 14.007/ 1.000* 1000) *100

= 0.535%

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Resistant and Non-resistant starch
A carbohydrate called resistant starch ferments in the large intestine while defying digestion in
the small intestine. The fibers function as a prebiotic and feed the beneficial bacteria in the gut as
they ferment. Resistant starch comes in a variety of forms. They are categorized according to
their source or structure.
Simply put, nonresistance carbohydrates are those that can be broken down by our digestive
enzymes. The carbohydrates are digested into glucose, which is our bodies' primary energy
source. This is crucial in moderation, but too much glucose leads to the production of fat that
isn't being utilised.
Non -resistant starch
Combine the supernantant solution obtained on centrifugation of the initial incubation. Decant
supernantant and resuspend the pellets in 2ml of 50% ethanol. Add a further 6ml of 50% solution
and carefully decant the supernantants and invert the tubes on absorbent paper to drain the excess
liquid and adjust the volume to 100 ml with 100ml sodium acetate buffer with a ph of 4.5 in a
volumetric flask. Mix well and incubate 0.1 aliquots of this solution in duplicates with 10
microlitre of the dilutre amg solution (300ul) in 100 mm sodium acetate buffer for 20mins at
50degrees.
Add 3.0 ml of GOPOD reagent and incubate the tubes at 50defgrees for 20 mins.
Do not use a vortex mixer to mix the contents as the starch may emulsify.

Resistant starch
 Accurately weigh 100 mg samples into each tube
 Add 4ml of pancreatic alpha amylase(10mg/ml) containing AMG (30/ml) to eacg of the
tubes and digest for 90mins.
 Incubate for 16hrs at 37degrees in a shaking water bath.
Add4ml of ethanol and shake thoroughly
 Centrifuge the tubes at 1500G for 10 mins
 Decant supernatants and resuspend then pellets into 2ml of 50% ethanol with vigorous
stirring. Add further 6ml of 50% ethanol solution. Mix the tubes.
 Centrifuge again at 1500G for 10 mins
 Decant supernatants and add 2ml of KOH to each tube. Put them in a water shaker for 20
mins
 Add 8ml of 1.2M sodium acetate buffer to each of the tube with stirring on the magnetic
stirrer. Immediately add 0.1M of AMG (solution A). place tubes in a water bath at 50
degrees for 30mins.

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 For samples containing >10% RS, qualitatively transfer contents to a volumetric flask.
Adjust to the 100ml mark and mix well.
 For samples containing <10% RS, directly centrifuge the tubes at 1500G for 10mins.
Transfer 0.1ml aliquots in duplicate of either into glass test tubes. Add 3ml of the
GOPOD reagent and incubate at 50 degrees for 20mins.
 Measure the absorbance of each at 510nm against the reagent of the blank


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 SOIL CHEMISTRY LABORATORY
KALRO's soil chemistry laboratory is a division of the department run by the lab staff. Its
primary duties include gathering and analyzing soil samples for pH, macronutrients, and
micronutrients, as well as providing advice to farmers on the best course of action to take during
farming. Other samples brought in for analysis in the lab include irrigation water, animal feed,
plant tissues, and manure.
The table that follows provides an overview of the various equipment used in the soil laboratory:
Instrument Function
Kjedahl Used in nitrogen distillation
Samples Plates Used to dry soil samples in the shade
Analytical balance Used to weigh the samples to be
analyzed
Water distiller Used to distill water
Kjedahl Used in nitrogen distillation
pH meter Used for soil PH determination
Glass wares Used in distillation process

Soil Analysis Protocol

The management of soil resources must include soil testing. Every sample that is taken needs to
be an accurate representation of the area being studied. The accuracy of the sampling determines
how useful the results of the laboratory analysis are.
In order to obtain a sample of the necessary size through subsampling, it is therefore advised to
gather a large number of samples. One sample is typically taken for every two hectares of land in
a given location. For a maximum area of five hectares, however, at least one sample must be
taken. In order to conduct a soil survey, samples are taken from a soil profile that is typical of the
soil in the surrounding area.

Soil Sample Preparation

When submitted to the soil laboratory, soil samples are gathered in sample plates or stacked in
trays before being given a special laboratory number. The soil submission form is then stamped
with the same laboratory number. The soils are then dried outside in the shade, away from the
sun, since light can influence the soil's water content and interfere with its structure. To reach the
requisite air-drying conditions, the samples are dried for 24 hours in the shade. After drying, soil
samples are taken.

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Soil PH Measurement
The inverse log of the hydrogen ion (H+) concentration is used to measure pH. The pH meter is
calibrated using several buffers with known pH values, such as buffer 4.0 and buffer 7.0 for
acidic soils, before analyzing the pH of the soil. Between PH of 4.0 to 5.5, micronutrients
including manganese, iron, zinc, boron, and copper are available to plants. The pH of the soil
solution determines the form and solubility of many plant nutrients. Nitrogen, potassium,
phosphorus, calcium, sulfur, and magnesium are made available to plants between the pH range
of 6.0 and 7.5. The pH analysis was conducted according to the following protocol:

are broken up with a mortar and pestle, then sieved through a 2-mm mesh sieve. After being
crushed, the samples are stored in various containers at the desired weight in grams for various
analyses.
• A soil sample of 10.0g was weighed, and 20ml of distilled water was added in a 1:2 ratio. After
stirring, the mixture was allowed to stand for 30 minutes. The pH of the soil sample was
measured after the pH meter had been calibrated.

CARBON ANALSIS
Colorimetric determination of organic carbon
Reagents
Barium chloride 0.4%, dissolve 4g barium Chloride in 100ml of DW
Potassium Dichromate 5%, dissolve 50g of potassium Dichromate in 1000ml of DW
H2SO4 Conc

Procedure
 Weigh 0.3 grams of soil in test tube
 Add 10ml of 5% potassium dichromate
 Add 5ml of H2SO4, Digest at 150degrees for 45 mins, allow to cool and add 50ml of
0.4% barium Chroride
 Swirl to mix and let to stand overnight
 Measure absorbance and standards of sample at 600nm
 Calculate concentration

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DETERMINATION OF EDTA-SOLUBLE CU, ZN, FE AND MN IN SOIL
Apparatus
Reciprocating shaker
AAS

REAGENTS
1%EDTA-= 10grams of di-sodium salt in 1000 of DW

Procedure
Weigh 10grams of soil in clean plastic bottle
Add 50ml of 1% EDTA
Shake for an hour
Filter, the supernatant is ready for analysis using AAS

DETERMINATION OF PHOSPHORUS BY DOUBLE ACID METHOD OF

EXTRACTION
Reagents
 Double acid (8.6ml HCL AND 1.4ml of H2SO4 IN 2000ml of DW)

Procedure
 Weigh 10 grams of the soil, add 50ml of double acid and shake for an hour
 Filter the supernatant and filtrate is ready for analysis using AAS.

DJEDAHL NITROGEN ANALYSIS

Reagents
 Preparation of the digestion mixture- measure 2.8grams of selenium powder, add 800ml
of conc. H2SO4, heat the solution until the original blackish color of selenium suspension
turns to green-blue to clear light yellow
 40% NAOH- dissolve 400g of NAOH pellets in 1000 of DW
 2% Boric acid- dissolve 20g of boric acid in 1000 DW

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  0.01N HCL – dilute 8.6ml of conc HCL in 1000ml, then dilute 100ml of that to 1000ml
mark with DW

Analysis
 Weigh 1g of soil, add 4ml of digestion mixture, digest at 110degrees for an hour and then
330 degrees for 2hours
 Add 25ml of DW then add 25ml of NaOH, distill while collecting the distillate in a
receiver (50ml conical flask) containing 25ml boric acid with drops of mixed indicator.
 Back titrate with 0.01M HCL

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 PATHOLOGY LABORATORY
Pathology is the study of diseases, including their causes, progression, and mechanisms. The
pathogens that cause various diseases include bacteria, fungi, viruses, and mycoplasma. These
diseases can spread through the air, soil, water, or seeds. Only virulent pathogens can actually
cause disease. Sap from the plant is removed using a sap extractor and then processed for
examination under a microscope in order to diagnose a viral pathogen-related plant disease.
Kenya Agricultural and Livestock Research's pathology lab deals with a variety of crop diseases
and numerous treatments for them. Its duties include enhancing crop quality and offering
farmers advice.
Its primary goal is to conduct disease diagnostics and pathogen identification for illnesses that
harm cash and food crops.
Farmers from a variety of plants may also submit diseased plant samples to the lab for
examination. Before analysis begins, the samples must be registered.
The majority of the research conducted at pathology labs involves field trials, established
experimental trials, and observations.

LABORATORY EQUIPMENT
All types of instruments, containers, and other equipment required for synthesis and analysis in
diverse laboratories are collectively referred to as "laboratory instruments." Laboratory
equipment must give accurate measurement findings, have a long lifespan, and ensure user
safety while being exposed to a variety of harsh chemical and physical forces. In order to fulfill
the high standards in laboratory technology, laboratory instruments must be of excellent quality
and durability.

INSTRUMENT FUNCTION
Compound microscope utilized to observe sample specimens
Flask shaker Used to mix samples vigorously for
homogenization and homogeneity.
Desiccator Applied to the quick drying of samples
Freezer Applied to the long-term, low-temperature
storage of samples for examination.
Laminar flow Used to disinfect work areas to remove
pathogens
Incubator Used to store samples for shorter periods of
time than a freezer at a temperature
between -4 and +4 degrees Celsius.
Stereo microscope Used to observe specimens in three

27
 dimensions.
Sap extractor Used to extract sap from plant samples that
have illness that is thought to be virus-
related.

WHEAT INNOCULATION
In order to keep the disease pressure high, wheat is vaccinated by introducing the pathogen that
causes stem disease into the crop. Stem rust disease spores are collected using a cyclone
machine, diluted in distilled water, filtered, and then injected using a needle and syringe into
several species of wheat plants. Inoculation techniques include spraying inoculum on leaves and
injecting inoculum into the stem of wheat using inoculation needles.
Dusting and the inoculum cyclone pump can both be used to apply inoculum to wheat fields.
Typically, different wheat cultivars are planted in distinct blocks. To enable investigation and
study of the various kinds' vulnerability to stem rust infections, experimental design is employed.

PLANY DIAGNOSTICS
Sometimes neither the indicators nor the symptoms are detailed or distinctive enough to
determine the etiology of an infectious plant disease. In some situations, it might be essential to
return a sample to the lab for additional testing to isolate and pinpoint the responsible agent. This
can be a labor- and time-intensive operation requiring specific knowledge.

EMBROGENESIS OF PLANT MATERIAL

A sample of the sick tissue was placed under conditions that would allow an infectious agent to
proliferate and maybe induce sporulation as one of the initial stages utilized in the lab. By
putting a leaf in a damp room, this was achieved. A sterile petri dish with a layer of wet filter
paper at the bottom of the dish, on top of which the sample was deposited, served as the moist
chamber. The main reason this kind of moist chamber is preferred is that it can accommodate
delicate and relatively flat objects like leaves.

FINDING AND IDENTIFYING THE CAUSAL AGENTS OF PLANT DISEASES

Pieces of infected plant tissue have to be placed on various nutritional medium in order to isolate
the fungi. When attempting to isolate the plant pathogen, several issues arose, but one was
particularly evident when using a dissecting microscope. Infected plant tissue contained more
saprophytes, which had moved into the infected tissue and outgrew the pathogen in terms of
nutrient medium, obscuring identification of the pathogen.

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MARKER ASSISTED SELECTION LABORATORY

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