THE KISUMU NATIONAL POLYTECHNIC

AN INDUSTRY SUCCESS

NAME: CAROLYNE OSIDE

COURSE: CERTIFICATE IN SCIENCE LABORATORY TECHNOLOGY

ADM NO: CSLT/M18/018

INDUSTRIAL ATTACHMENT REPORT

2019

i
DECLARATION
This research report is my original work and has not been presented for certificate award in
any other institution.

Signature…………………………………………… Date……/……/……………

OSIDE CAROLYNE

CSLT/M18/018

This report has been submitted for Research with my approval as the institution Supervisor.

Signature…………………………………………… Date……/……/……………

Kenya Agricultural and Livestock Research Organisation P. O. Box Private Bag 20107,
Njoro.

ii
DEDICATION
This dissertation is dedicated to my family for their constant support during its compilation
and to Kenya Agricultural and Livestock Research Organisation staff for equipping me
properly for the necessary skills.
Thank you and God bless you.

iii
ACKNOWLEDGEMENT
Glory to God for the gift of good health, knowledge and understanding during my attachment
period.
My sincere gratitude goes to JKUAT Medical Microbiology Department for equipping and
nurturing me with the necessary skills that has made it possible for my successful attachment.
I acknowledge my supervisor Dr. Edna Songoro for having made it possible to supervise me
on time.
Am so much grateful to KALRO-Njoro staff as a whole, they were able and willing to guide
me throughout my attachment. I also appreciate my able and hardworking industrial
supervisor Mr Kimani Ndung’u; my institution supervisor for continued guidance and
assistance during this period. Mr Kimani exposed me to the practical aspects of my degree
Programme. He has been a mentor and a source of inspiration to me, his dedication to teach
me allowed me to acquire and develop valuable technical, professional and practical skills. I
do appreciate all the support, motivation and guidance he gave me during this period.

I would also like to express my sincere gratitude to my loving family for their emotional and
financial support; they have been a great source of encouragement throughout my attachment
period. Finally to my friends and colleagues whom with their love, support, understanding
and patience made my stay at KALRO-Njoro enjoyable, thank you so much. God bless you
all

iv
ABBREVIATIONS AND ACRONYMS
KALRO-Njoro – Kenya Agricultural and Livestock Research Organisation-Njoro

FCRI-Food Crop Research Institute

PCR-Polymerase Chain Reaction

NGOs-Non-Governmental Organizations

ICIPE - International Centre of Insect Physiology and Ecology

EASEED- East African Seed Co. Ltd -Company Directory

AFFA- Agriculture, Fisheries and Food Authority

NASECO- NALWEYO SEED Company

FBOs- Farmer Based Organization

FIPs- Farm Input Promotions

KEPHIS- Kenya Plant Health Inspectorate Service

ADC- Agricultural Development Corporation

vi
LIST OF FIGURES
Figure 1: DNA Prepared samples 12

Figure 2: DNA samples in a Gel 12

Figure 3: Nucleic acid in a UV Trans illuminator 13

Figure 4: PCR stages in a thermo cycler 18

Figure 5: Disease triangle 21

Figure 6: Stem rust on barley 23

vii
LIST OF TABLES
Table 1: KALRO Centers 6

Table 2: Genetic markers 19

viii
LIST OF EQUATIONS
Equation 1: Digestion process of Nitrogen 25

Equation 2: Distillation process 25

Equation 3: Titration 26

ix
 TABLE OF CONTENTS
DECLARATION...................................................................................................................................ii
DEDICATION......................................................................................................................................iii
ACKNOWLEDGEMENT....................................................................................................................iv
ABBREVIATIONS AND ACRONYMS.............................................................................................vi
LIST OF FIGURES.............................................................................................................................vii
LIST OF TABLES..............................................................................................................................viii
LIST OF EQUATIONS........................................................................................................................ix
ABSTRACT (SUMMARY).................................................................................................................xi
CHAPTER ONE....................................................................................................................................1
INTRODUCTION.................................................................................................................................1
1.1 Geographical Location.................................................................................................................1
1.2 Historical Background.................................................................................................................1
1.3 Mission Of The Organisation.......................................................................................................1
1.4 Vision..........................................................................................................................................1
1.5 Objectives....................................................................................................................................1
1.6 Mandate.......................................................................................................................................1
1.6.1 Technology Development & Adaptation Of:............................................................................1
1.6.2 Additional Satellite Services.....................................................................................................2
1.7 Core Values.................................................................................................................................3
1.8 Core Functions.............................................................................................................................3
1.9 Organizational Structure..............................................................................................................8
1.10 Fcri Institute Programmes Objectives........................................................................................8
1.11 Institute Clients..........................................................................................................................9
1.12 Institute Partners / Stakeholders/ Collaborators.........................................................................9
CHAPTER TWO.................................................................................................................................10
LABORATORY ROLES AND ACTIVITIES....................................................................................10
2.0 Introduction...............................................................................................................................10
2.1 Biotechnology Laboratory.........................................................................................................10
2.1.1 DNA Extraction......................................................................................................................10
2.1.1.1 CTAB Protocol............................................................................................................10
2.1.1.2 Gel Electrophoresis..........................................................................................................11
2.1.1.3 Sodium Dodecyl Sulphate (SDS) Protocol.......................................................................15
2.1.1.4 Nucleon Phytopure Kit....................................................................................................16
2.1.1.5 Polymerase Chain Reaction.............................................................................................17

x
 2.1.1.6 Molecular markers...........................................................................................................19
2.2 Pathology...................................................................................................................................21
2.3 Soil Chemistry...........................................................................................................................23
2.3.1 Soil tests.................................................................................................................................23
2.3.1.1 Soil sampling...................................................................................................................24
2.3.1.2 Soil pH analysis...............................................................................................................24
2.3.1.3 Nitrogen Analysis (Kjeldahl method)..............................................................................25
2.3.1.4 Determination of EDTA soluble; copper, zinc, iron and manganese in the soil Preparation
of 1% EDTA................................................................................................................................27
2.3.1.5 Determination of sodium, potassium, calcium, magnesium and phosphorous by Double
Acid method of extraction...........................................................................................................27
CHAPTER THREE.............................................................................................................................28
CONCLUSION AND RECOMMENDATION...................................................................................28
3.1 Conclusion...........................................................................................................................28
3.2 Recommendation.................................................................................................................28
REFERENCE......................................................................................................................................29

xi
ABSTRACT (SUMMARY)
Some of the activities carried out at the biotechnology laboratory included but not limited to
DNA extraction by CTAB Protocol, Gel electrophoresis, DNA extraction by Modified CTAB
Protocol, Sodium Dodecyl Protocol and Nucleon Phytopure Kit, Tissue culture, Polymerase
Chain Reaction, Molecular Plant Pathology and Molecular Markers.

In the soil chemistry, the skills learned included soil sampling, soil pH, Kjeldahl Nitrogen
Method, Carbon analysis, Determination of EDTA Soluble ;Copper, Zinc, Iron and
Manganese in the soil, Determination of Sodium ,Potassium, Calcium, Magnesium and
Phosphorous by Double Acid Method of Extraction, Calorimetric determination of
Phosphorous and recommending to farmers who wants to plant a certain crop.

In the pathology laboratory, how to identify plant diseases, meteorology and incubation and
microscopy were taught.

This report therefore gives a conceptual view of the various labs I was attached in as well as
the duties I undertook during the attachment period. It begins with a detailed walkthrough on
the background of KALRO-Njoro, which is an acronym for Kenya Agricultural and
Livestock Research Organisation. Then it gives an account of the specific areas that I went
through. It also presents the experience I encountered during the attachment period as well as
the relevance of the attachment to my course. It finally gives the conclusion and
recommendation.

xii
 CHAPTER ONE
INTRODUCTION

1.1 Geographical Location

The Kenya Agricultural and Livestock Research Organization (KALRO), Food Crops
Research Centre Njoro is located 20 Km South West of Nakuru town in Western rim of Rift
Valley. It's in Njoro Sub-County of Nakuru County 200 Km from Nairobi at an altitude of
2120 meters above sea level.

1.2 Historical Background

KALRO Njoro has a long history of food crops research, it is one of the oldest in the country.
It was established in 1927 for the management of wheat rust diseases, following introduction
of wheat in the country earlier in the century. Since then, the Centre has steadily grown to
host a world class level phenotyping platform for the rust diseases and has expanded its
research components to include research in oil crops, in tubers and roots and in agronomy,
soil and water management, integrated pest and disease management, and socio-economics.

1.3 Mission Of The Organisation

To generate and disseminate food crops' knowledge, innovative technologies and services
that respond to clientele demands for sustainable livelihoods in Kenya

1.4 Vision
To be a global competitive agricultural Institute in Food Crops Research

1.5 Objectives
i. To generate and promote food crop technologies.
ii. To develop and promote markets and marketing strategies for food crop product
chains.
iii. To facilitate and advocate policy option for enhancing demand-driven food crop
product value chains.
iv. To strengthen the capacity for implementing food crop value chains research.
v. To enhance availability of knowledge, information and technologies on food crop
product value chain research.

1.6 Mandate
KALRO Food Crops Research Institute have the mandate to develop and promote staple food
crops technologies in 47 counties of Kenya. To undertake this task seven FCRI centers are
responsible that is KALRO ECRI Kitale, KALRO FCRI Njoro, KALRO FCRI Embu,
KALRO FCRI Alupe, KALRO FCRI Kisii, KALRO FCRI Muguga, KALRO FCRI Kabete

1.6.1 Technology Development & Adaptation Of:

 Cereal crops

 Root & Tuber

1
 Legume crops

 Natural resources management

 Crop protection

 Food science

 Socio-economics

 Technology transfer

 Analytical Soil Laboratory services

 Analytical Pathology Services

 Analytical Insect identification

 Analytical mycotoxin assessment

1.6.2 Additional Satellite Services

 Livestock management

 Vegetable management

 Fruit tree production

2
1.7 Core Values
 Scientific Excellence

 Collaboration

 Creativity and innovation

 Self-respect and discipline

 Transparency and accountability

 Integrity

 Impact performance and service delivery oriented

 Consultativeness

  Respect for client confidentiality

 Implementation of platforms

1.8 Core Functions

 Basic and Strategic research on cereal crops, grain legumes and root and tuber crops,
biotechnological approaches natural resource management socioeconomics and
research methods and postharvest and food nutrition and processing

 Technology development, adaptation and promotion

 Partnerships with other agricultural sector ministries and relevant partners

 Contribute towards attaining self-sufficiency in animal food and non-food products to

meet the national demand and create gainful employment;

 Contribute towards increasing household income and hence standard of living of

resource-poor pastoralist farmers by developing appropriate livestock production
technologies;

 Dissemination of research technologies in collaboration with extension staff of the

Ministries of Agriculture, Livestock and Fisheries Development through
demonstrations, field days, shows, workshops, seminars, training and visits;

 Establish a strong farmer-research-extension linkage to facilitate technology

development and dissemination;

 Contribute to KALRO self-reliance through generation of Appropriation in Aid

(AIA);

 Capacity building among farmers, students and other stake holders;

3
 Establish partnerships with agro-based industries, consumer organizations, donors and
non-governmental organizations;

 Establish techno-farms in the centers as a training tool for farmers, students and other
stakeholders;

 Develop capacity for information and knowledge acquisition, storage and

dissemination.

 Problem identification, prioritization and generation of appropriate research

interventions;

KALRO Institutes and Centres

The Kenya Agricultural and Livestock Research Act, 2013, establish sixteen (16) Institutes
within the KALRO and 47 Centres/Sub-Centres as per the table below:

NO INSTITUTE CENTER Status

Thika Headquarters

Tigoni

1 Horticulture Research Institute

Matuga

Kibos

4
3 Apiculture Research Institute Perkerra Headquarters

Arid and Range Lands Research

4 Kiboko Headquarters
Institute

Lanet

Mariakani
5 Beef Research Institute

Garissa Headquarters

Trans mara

Biotechnology
6 Biotechnology Research Institute

Muguga Headquarters

Msabaha

7 Dairy Research Institute Ol joro orok

Naivasha Headquarters

Kitale Headquarters

Katumani

Muguga

5
 Kabete

8 Food Crops Research Institute

Njoro

Embu

Kisii

Alupe

9 Genetic Resources Research Gene bank Headquarters

Centre

Mwea-tabere

10 Industrial Crops Research Institute Nprc molo

Mtwapa

11 Non-Ruminant Research Institute Kakamega Headquarters

Marsabit Headquarters
12 Sheep and Goat Research Institute

Buchuma

13 Veterinary Research Institute Muguga Headquarters

Alupe

6
14 Tea Research Institute Kericho Headquarters

Kangaita

Kibos Headquarters

Opapa

Mumias

15 Sugar Research Institute

Mtwapa

Nyando

Kikoneni

Ruiru Headquarters

Mariene

Namwela

16 Coffee Research Institute Kisii

Kitale

Koru

7
 Azania

Table 1: KALRO Centres

1.9 Organizational Structure

 KALRO FCRI- operates under Institute Director. The Institute Director is supported
by seven Centre Directors Centre who oversee various programmes cereal crops, grain
legumes and root and tuber crops, natural resource management, socioeconomics and
research methods and postharvest and food nutrition and processing

1.10 Fcri Institute Programmes Objectives

 To develop, disseminate and promote cereal crops (maize, wheat, sorghum millets and
grain amaranth) varieties, their seed production and attendant integrated crop management
packages suitable all ecozones of Kenya.

 To develop, disseminate and promote root and tuber crop (sweet potato, cassava,
potato, yam, arrow root) varieties, their seed conservation and attendant integrated crop
management packages suitable all ecozones of Kenya.

 To develop, disseminate and promote legume grain crops (beans, peas, soya bean)
varieties and attendant integrated crop management packages suitable all Eco zones of
Kenya.

 To develop, validate, disseminate and catalyse the adoption of postharvest and food
nutrition technologies suitable for enhancing the health of households of various Eco zones.

 To develop, validate, disseminate and catalyse the adoption of sustainable Natural

resource management technologies (carbon credits, minimum and zero tillage, rotational 4
agriculture, soil and water management, suitability mapping) in the different Eco zones of
Kenya.

 To develop, validate, disseminate and catalyse the adoption of improved crop

protection technologies responding to dynamic surges of invasive and emergent insect
(millipede epidemics, army worms, locusts), disease (MLN, wheat stem rust), birds (quelea
quelea) and weed ( pests vibrantly, innovatively through monitoring and surveillance and
affective and action based approaches.

 To link biophysical research to stakeholders for socio-economic understanding of

farming systems, prioritization of constraints and documentation of impacts. To catalyse,
disseminate and promote FCRI developed technologies in partnership with stakeholders.

1.11 Institute Clients

Farmers, traders, agrochemicals companies, Schools colleges, Universities, Seed Companies,
farmers of neighboring counties like Uganda, Tanzania, Ethiopia other KALRO Institutes and
International Research centres, International farmers and other communities.

8
1.12 Institute Partners / Stakeholders/ Collaborators
 Govt. Ministries: MoAL&F, MoE&WR, Ministry of Education, Science and
Technology, Ministry of Labour, Social Security and Services, AFFA  NGOs, FBOs (FiPs,
Leldet, AGRA

 Seed Companies: Kenya Seed, Unga Ltd, FRESHCO, EASEED, SEEDCO,

NASECO, Monsanto, Western Seed, ROTAM, Bubayi Seed, Dryland Seed, Western Seed
company)

 Parastatals: KEPHIS, Kenya seed company, ADC, Moi University, JKUAT

University, University of Eldoret, Mount Kenya University and Kenyatta University

 CGIAR/other Research Institutions: CIMMYT, CIAT, ICRISAT, ICIPE, CIP,

ASARECA,

 International Research Organization (AGRA)

9
 CHAPTER TWO
LABORATORY ROLES AND ACTIVITIES

2.0 Introduction
This chapter discusses all the activities carried in the following laboratories; Biotechnology
laboratory, Soil Chemistry laboratory and Pathology laboratory.

2.1 Biotechnology Laboratory

Biotechnology is the exploitation of biological processes for industrial and other purposes,
especially the genetic manipulation of microorganisms for the production of antibiotics,
hormones, etc. it involves living systems and organisms to develop or make products, or "any
technological application that uses biological systems, living organisms, or derivatives thereof,
to make or modify products or processes for specific use.

2.1.1 DNA Extraction

DNA extraction is the technique used to isolate DNA in a biological sample. DNA Extraction is
extracting DNA from the cell by disrupting the cell wall/ cell membrane and nuclear membrane
with the help of the chemical, physical or mechanical methods

The ability to extract DNA is of primary importance to studying the genetic causes of disease
and for the development of diagnostics and drugs. It is also essential for carrying out forensic
science, sequencing genomes, detecting bacteria and viruses in the environment and for
determining paternity.

There are various protocols used in DNA Extraction. These includes;

1. CTAB Protocol

2. Modified CTAB Protocol

3. Sodium Dodecyl Sulphate (SDS) Protocol

4. Nucleon Phytopure Kit

2.1.1.1 CTAB Protocol

CTAB DNA extraction buffer is more suitable for extracting DNA from the plant tissues.
Because of the high content of the secondary metabolites, proteins, polysaccharides and
polyphenolic compounds into the plant cell, it is very difficult to extract DNA.

CTAB DNA extraction buffer facilitates DNA isolation from harder tissues such as plant cells.

Components of CTAB buffer

I. CTAB detergent-Dissolves liquid and unwinds DNA from chromatinprotein.

II. DD H2O-It’s a universal solvent and a reaction media.

III. EDTA-It’s a chelating agent; it binds cations, prevents actions of coenzymes.

10
V. Na2SO3, PVP-Antioxidants;Prevent oxidation of the sample VI. Tris-HCL-Is a buffer;
stabulises the pH

CTAB Protocol procedure

I. 0.3g of the leaf sample was crushed using a mortar and pestle

II. 800µl of CTAB buffer was added to the sample before and after crushing

III. The sample was then placed in a 2ml microcentrifuge tube

V. It was mixed by inversion 7 times

VI. It was centrifuged at 13000rpm for 10 minutes

VII. 850µl of the liquid phase was pipetted out into a new 2ml microcentrifuge tube
VIII. The remainder was discarded in a discarding jar
IX. 850µl of Chloroform Isoamyl Alcohol(CIA) was then added
X. It was mixed by inversion until the solution was milky

XI. It the then centrifuged at 13000rpm for 10 minutes

XII. 750µl of the upper layer(aqueous phase) was pipetted into a 1.5ml microcentrifuge tube
XIII. The remnants were discarded off
XIV. 750µl of ice cold isopropanol was added
XV. It was then gently inverted and the mucoid liquid observed
XVI. It was incubated at -20ºc for 40 minutes
XVII. Then it was centrifuged at 13000rpm for 10minutes
XVIII. The liquid phase was gently poured off
XIX. 600µl of 70% ice cold ethanol was added
XX. It was centrifuged at 13000rpm for 10 minutes XXI. 70% ethanol was poured off

XXII. It was air-dried for 30 minutes to enable evaporation of 70% ethanol and remain with
nucleic acid pellet
XXIII. 50µl of DD H2O was added
XXIV. It was suspended in a water bath shaker for 30 minutes.

2.1.1.2 Gel Electrophoresis

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins
according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by
an electrical field through a gel that contains small pores. The molecules travel through the pores
in the gel at a speed that is inversely related to their lengths. This means that a small DNA
molecule will travel a greater distance through the gel than will a larger DNA molecule.

11
Components of Gel electrophoresis

The components of gel electrophoresis system are the following:

 A Slab holder for vertical or horizontal gels (thin, flat sheets of many individual lanes)
 Polyacrylamide or agarose gels (cm x cm x mm); these are poured for each analysis 
Gel is amended with SDS to dissociate & charge proteins.  High voltage power supply
(0.1-6 kV)
 A detection technique (dye staining, fluorescence, or autoradiography to image separated
bands)

Procedure of Gel electrophoresis

I. 1g of Agarose was weighed and put in a volumetric flask

II. 100ml of Sodium Borate Buffer was added and placed in the microwave for 2minutes at
650 watts

III. 3µl of Ethidium bromide was added(in the fume hood)

V. It was poured off to the gel tray VI. The gel comb was then inserted

VII. It was then left for 30 minutes to cast

12
Gel preparation

Prepared samples are pipetted into each of the wells of the gel/slab holder.

Figure 1: DNA Prepared samples

Electrodes are already plumbed into the gel holder above and below the gel and they contact
the gel via a liquid buffer. The connectors are electrically isolated from each other but contact
opposite edges of the gel.

Figure 2: DNA samples in a Gel

Separation

Running the gel involves connecting the electrodes to the power supply. Heat is generated
during the run and passively dissipates or is removed via (a water-powered) cooling chamber
Sandwiched against the gel. Higher current generates more heat.

13
Figure 3: Nucleic acid in a UV Transilluminator

After the run, these bands can be examined only after an appropriate dye or
imaging/development technique is used. A tracking dye can be added in the buffer to
visualize the mobility front to help decide when to stop the run. If pre-stained standards are
used their bands can be seen as the electrophoresis proceeds.

In this form of gel electrophoresis, the proteins are all of equal charge/mass, so separation is
based solely on protein mass as they migrate through the gel under the electrical potential.

Nucleic acid component

5µl of nucleic acid and 3µl sample loading buffer (enhances density of the Nucleic Acid
sample and enables visual tracking of the DNA migration) Components of the sample
loading buffer

I. DD H2O-Universal solvent

II. Buffer-Resist changes to pH

III. Glycerol or sucrose or Ficoll-400-Enhances density

IV. Dye-for visual tracking of the DNA migration.

14
2.1.1.3 Sodium Dodecyl Sulphate (SDS) Protocol
SDS Protocol procedure

I. 0.3g of the leaf sample was crushed using a mortar and pestle

II. 800µl of the Sodium Dodecyl Protocol buffer was added to the sample before and after
crushing

III. The sample was then placed in a 2ml microcentrifuge tube

V. It was mixed by inversion 7 times

VI. It was centrifuged at 13000rpm for 10 minutes

VII. 850µl of the liquid phase was pipetted out into a new 2ml microcentrifuge tube
VIII. The remainder was discarded in a discarding jar
IX. 850µl of Chloroform Isoamyl Alcohol(CIA) was then added
X. It was mixed by inversion until the solution was milky

XI. It the then centrifuged at 13000rpm for 10 minutes

XII. 750µl of the upper layer(aqueous phase) was pipetted into a 1.5ml microcentrifuge tube
XIII. The remnants were discarded off
XIV. 750µl of ice cold isopropanol was added
XV. It was then gently inverted and the mucoid liquid observed
XVI. It was incubated at -20ºc for 40 minutes
XVII. Then it was centrifuged at 13000rpm for 10minutes
XVIII. The liquid phase was gently poured off
XIX. 600µl of 70% ice cold ethanol was added
XX. It was centrifuged at 13000rpm for 10 minutes XXI. 70% ethanol was poured off

XXII. It was air-dried for 30 minutes to enable evaporation of 70% ethanol and remain with

XXIII nucleic acid pellet50µl of DD H2O was added

15
2.1.1.4 Nucleon Phytopure Kit.
Reagents

I. Reagent 1

II. Reagent 2

III. Resin(Phytopure resin)

IV. Chloroform Procedure

I. 0.3g of the leaf material was crushed after adding 600µl of reagent 1

II. 200 µl of reagent was added.

III. It was then placed in a 2ml microcentifuge tub

V. It was incubated at 65º C for 10 minutes

VI. It was placed on ice for 20 minutes

VII. 500 µl of chloroform was added
VIII. 100 µl of phytopure resin was added then shaken vigorously
IX. It was then placed on atilt shaker for 20 minutes at a room temperature
X. It was centrifuged at 1000rcf for 7 minutes XI. 600 µl was pipetted out

XII. 600 µl of ice-cold isopropanol was added

XIII. Mucoid-like feature was then observed
XIV. It was then incubated for 20 minutes at -20ºC
XV. It was centrifuged at 11000rcf for 7 minutes
XVI. The liquid phase was poured off carefully
XVII. 600 µl of ice cold 70% ethanol was added
XVIII. It was centrifuged at 11000rcf for 7 minutes
XIX. The ethanol was poured off
XX. It was air dried to remove the residual 70% ethanol XXI. 50 µl of ultrapure water (DD
H2O) was added

XXII. It was then incubated at 65ºC for 20 minutes in a water bath shaker to enable dissolving
of nucleic acid pellet

NB: to remain with pure DNA solution, add 2µl per 50 µl RNase A enzyme

16
2.1.1.5 Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a method widely used in molecular biology to make
several copies of a specific DNA segment. Using PCR, copies of DNA sequences are
exponentially amplified to generate thousands to millions of more copies of that particular
DNA segment.

Components of PCR

I. DD H2O-Universal solvent

II. Buffer-resist changes in pH

III. Magnesium Chloride-enhances reaction of ions and co-ions

V. Template-specific DNA sequence acted upon during the reaction(contains target DNA

sequence to be amplified)

VI. Primer-this are short oligonucleotides strands that are complimentary to the target
sequence DNA template

VII. Deoxynucleotide tri-phosphates-adds nucleotides to the strand, provide energy to the

reaction

Stages

Initialization: It consists of heating the reaction chamber to a temperature of 94–96 °C

Denaturation: This step is the first regular cycling event and consists of heating the reaction
chamber to 94–98 °. This causes DNA melting, or denaturation, of the double-stranded DNA
template by breaking the hydrogen bonds between complementary bases, yielding two single-
stranded DNA molecules.

Annealing: In the next step, the reaction temperature is lowered to 50–65 °C for 40 seconds,
allowing annealing of the primers to each of the single-stranded DNA templates. Two
different primers are typically included in the reaction mixture: one for each of the two
single-stranded complements containing the target region. The primers are single-stranded
sequences themselves, but are much shorter than the length of the target region,
complementing only very short sequences at the 3' end of each strand.

It is critical to determine a proper temperature for the annealing step because efficiency and
specificity are strongly affected by the annealing temperature. This temperature must be low
enough to allow for hybridization of the primer to the strand, but high enough for the
hybridization to be specific, i.e., the primer should bind only to a perfectly complementary
part of the strand, and nowhere else. If the temperature is too low, the primer may bind
imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature
is about 3–5 °C below the Tm of the primers used. Stable hydrogen bonds between
complementary bases are formed only when the primer sequence very closely matches the
template sequence. During this step, the polymerase binds to the primer-template hybrid and
begins DNA formation.
17
Extension/elongation: The temperature at this step depends on the DNA polymerase used;
the optimum activity temperature for the thermostable DNA polymerase of Taq (Thermus
aquaticus) polymerase is commonly 72 °C, the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template strand by adding free dNTPs from the reaction
mixture that are complementary to the template in the 5'-to-3' direction, condensing the 5'-
phosphate group of the dNTPs with the 3'-hydroxy group at the end of the nascent
(elongating) DNA strand.

Final elongation: This single step is performed at a temperature of 70–74 °C (158–165 °F)
(the temperature range required for optimal activity of most polymerases used in PCR) for 5–
15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully
elongated.

Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite
time, and may be employed for short-term storage of the PCR

18
Steps in the thermocycler

Final denaturation

Initial extension

Final extension

Annealing
Initial denaturation
Figure 4: PCR stages in a
thermocycler

To check whether the PCR successfully generated the anticipated DNA target region (also
sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis may be
employed for size separation of the PCR products. The size(s) of PCR products is determined
by comparison with a DNA ladder, a molecular weight marker which contains DNA
fragments of known size run on the gel alongside the PCR products.

N/B: Utmost care must be taken when handling ethidium bromide and when working with
U.V light. Protective clothing and masks must be worn at all time.

2.1.1.6 Molecular markers

Molecular marker is a biotool consisting of specific and unique sequences that are used to
identify gene/chromosomal regions and positions; and differentiate between two distinct
DNA sequences (polymorphism)

19
Types of genetic markers

List of Markers Acronym

Restriction Fragment Length Polymorphism RFLP

Random Amplified Polymorphic DNA RAPD

Amplified Fragment Length Polymorphism AFLP

Variable Number Tandem Repeat VNTR

Oligonucleotide Polymorphism OP

Single Nucleotide Polymorphism SNP

Allele Specific Associated Primers ASAP

Inverse Sequence-tagged Repeats ISTR

Inter-retrotransposon Amplified Polymorphism IRAP

Table 2: Genetic markers

20
2.2 Pathology
Pathology is an area which includes a number of distinct but inter-related medical specialties
that diagnose disease, mostly through analysis of tissue, cell, and body fluid samples. It is the
predicted or actual progression of particular diseases.

2.2.1 Field and Molecular plant pathology

Plant diagnostic-This is the identification/screening of an illness/infection caused by a

pathogen or abiotic factors by looking at signs and symptoms. This can be done by the
following ways:

I. Optical/visual-This is the identification of affected plants and recognising the

healthappearance.This can also be done through microscopy.

II. Molecular-This can be done through PCR.

III. Biochemical-through growing it in culture media.

IV. Serology-This can be done by testing antibody-antigen reaction by Enzyme Linked

Immuno-Sorbent Assay (ELISA).

2.2.2 Disease Triangle

This shows the interrelationship between the host, environment and pathogen causing an
infection.

The disease triangle is a fundamental principle illustrating the factors involved in the
occurrence and severity of plant disease. Disease caused by a living agent requires the
interaction of a susceptible host, a virulent pathogen, all in the context of a favourable
environment. Plant disease is prevented when any one of these three components are
eliminated. The three factors can be manipulated to reduce the risk of disease development

Some plant pathologists have expanded the usefulness of the disease triangle concept by
adding one or more factors such as “human activity” or “vectors” to represent special-case
applications. Another useful factor is “time” and it can be represented by one of the vertices
of a three-dimensional pyramid (here with the top of the pyramid pointing out at you).

21
Figure 5: Disease triangle

2.2.3 Koch’s postulates

In 1890 the German physician and bacteriologist Robert Koch set out his celebrated criteria
for judging whether a given bacteria is the cause of a given disease:

 The bacteria must be present in every case of the disease.

 The bacteria must be isolated from the host with the disease and grown in pure
culture.

 The specific disease must be reproduced when a pure culture of the bacteria is
inoculated into a healthy susceptible host.

 The bacteria must be recoverable from the experimentally infected host.

22
2.2.4 Commonly Observed Disease Signs and Symptoms

I. Fungal leaf spots - spots usually vary in size. Generally are round and occasionally
elongate on stems. Zones of different colour or texture may develop giving the spot a "bull's
eye" effect. Spots are not limited by leaf veins.

II. Bacterial leaf spots - spots are often angular due to limitation by leaf veins. Colour is
usually uniform and no signs of plant pathogen are evident. Tissue may appear initially as
being water soaked but may become papery as it dries.

III. Mosaic and Ringspot - Mosaic (Figure 9) and ringspot are used to describe an

irregular patchwork of green and yellow areas over the surface of a leaf. In some cases leaves
may also become distorted. Often these symptoms are associated with viral pathogens. There
is not a sharp margin between the affected and healthy areas. Distinct margins may indicate a
nutritional problem or genetic variegation.

IV. Presence of Spores/Spore Structures - Several fungal diseases can be easily

identified based on the presence of spores or spore structures on the leaf surface. Some
examples of this are rusts which are often recognized by the rusty brown to black spores.

Figure 4. Stem rust on barley. Caused by

Puccinia graminis. (Courtesy B.Steffenson)

Figure 6: Stem rust on barley

2.3 Soil Chemistry

Soil chemistry majorly focuses on the chemical reactions in soils that affect plant growth and
plant nutrition. Soil chemistry is the study of the chemical characteristics of soil. Soil
chemistry is affected by mineral composition, organic matter and environmental factors.

2.3.1 Soil tests

This is the analysis of soil samples to determine the nutrient and contamination levels,
composition and other characteristics like acidity and other nutrient composition. These tests
include:

I. pH analysis

II. Nitrogen analysis

III. Carbon analysis

23
IV. Determination of EDTA soluble; copper, zinc, iron and manganese in soil

V. Determination of sodium, potassium, calcium, magnesium and phosphorous by

Double Acid method of extraction

VI. Calorimetric determination of Phosphorous

2.3.1.1 Soil sampling

Representative soil samples should be recovered, processed and labeled before analysis.

Several samples should be collected across the field, bulked, and mixed and a representative
soil sample recovered.

How to sample the soil

Equipment used

A soil augur

How to sample

For three crops (coffee, fruit trees)

Samples were taken to a depth of 60cm at 20cm increment that is 0-20cm, 20-40cm, and 40-
60cm using a soil augur.

For shallow rooted crops (maize, beans, potatoes) Samples were taken to a depth of 20cm
using a soil augur.

2.3.1.2 Soil pH analysis

Soil pH is a measure of the acidity and alkalinity in soils. pH levels range from 0 to 14, with
7 being neutral, below 7 acidic and above 7 alkaline. The optimal pH range for most plants is
between 5.5 and 7.0; however, many plants have adapted to thrive at pH values outside this
range. Because pH levels control many chemical processes that take place in the soil –
specifically, plant nutrient availability – it is vital to maintain proper levels for your plants to
reach their full yield potential.

Procedure

I. The samples were dried to overcome eventual decomposition processes which may
change the original chemical composition.

II. The soil sample was crushed using a pestle and a mortar.

III. It was then sieved through a 2mm sieve.

IV. The pH was calibrated using buffer 4 and buffer 7 for acidic soils and buffer 7 and
buffer 9 for basic soils.

V. 10g of air-dried soil was weighed.

VI. 20ml of distilled water was added and stirred well then left to stand for 30 minutes.
24
VII. pH readings were then taken using a pH meter.

NB: Soil pH can be lowered by adding gypsum (calcium sulphate dihydrate) if it is too
alkaline or increased by adding limestone (calcium carbonate) if it’s too acidic.

2.3.1.3 Nitrogen Analysis (Kjeldahl method)

The process involves three steps:

I. Digestion

II. Distillation

III. Titration

Digestion

1g of the soil sample was weighed into a clean digestion tube.

4ml of the digestion mixture was added.

It was then digested at 110ºc for 1hour and at 330ºc for 2hours and allowed it to cool.

Equation 1: Digestion process of Nitrogen

Organic N H2SO4 →

(NH4)SO4 H2O CO4 other sample matrix byproducts

Distillation

25ml of distilled water was added to the digested sample.

25ml of sodium hydroxide was then added to the sample (this converts ammonium to
ammonia)

Equation 2: Distillation process

ammonium ammonia

heat

sulfate gas

(NH4)2SO4 2NaOH → 2NH3 Na2SO4 2H2O

25
.25ml of boric acid and 3 drops of mixed indicators was added into a separate conical flask
(boric acid is an absorbing solution).

The ammonia is qualitatively captured by the boric acid solution forming solvated ammonia
ions.

The distil and the distillate is then collected in the receiver conical flask.

Titration

The solution was the titrated using 0.01MHCL.

Equation 3: Titration

standard excess
sulfuric
Ammonia sulfuric acid ammonium
acid
acid sulfate

2NH3 2H2SO4 → (NH4)2SO4 H2SO4

(no color change)

measured measured
ammonia ammonium
excess sodium
sulfate sulfate
acid hydroxide

(NH4)2SO4 H2SO4 2NaOH → Na2SO4 (NH4)2SO4 2H2O

26
 (color change occurs)

Formulae of calculating % Nitrogen

%Nitrogen=(T − B) × N × AW⁄M × 1000 × 100%

2.3.1.4 Determination of EDTA soluble; copper, zinc, iron and manganese in the soil
Preparation of 1% EDTA
10g of Disodium salt in 1000ml of distilled water.

Procedure

10g of the soil sample was weighed into a clean plastic bottle.

50ml of 1% EDTA was added and then shaken for 1 hour.

It was filtered and the filtrate was analyzed using Atomic absorption spectrophotometer for
copper, zinc and manganese

2.3.1.5 Determination of sodium, potassium, calcium, magnesium and phosphorous by

Double Acid method of extraction
Double acid preparation

8.6ml HCL and 1.4ml of sulphuric acid was added in 2000ml of distilled water.

Procedure

10g of the soil sample was weighed then 50ml of double acid added It was shaken for 1 hour
then filtered.

The filtrates were ready for analysis using atomic absorption spectrophotometer for sodium,
potassium, calcium, magnesium and ultraviolet spectrophotometer for phosphorus.

27
 CHAPTER THREE
CONCLUSION AND RECOMMENDATION

3.1 Conclusion
KALRO has helped me to relate my academic work with practical fieldwork by accessing
various activities done by applying microbiological techniques and related fields. My
interrelationship with the KALRO staff is of great importance and cannot be overlooked
because it has improved my team experience and equipped me with regards to performance
and knowledge since it has acted as a bridge between theory and practical. The research
activities carried out at KALRO has enabled me to gain a lot of knowledge and skills that is
very much related to my course. The skills acquired will help me to better my career and help
in the various community services at large. I would like to encourage students from the
university to apply for places of attachment in this institution and use the available
opportunities to better their skills. In general, the attachment was very skillful and full of
experience.

3.2 Recommendation
Despite the fact that KALRO-Njoro has had a long and good history as a convenient center
for student attachment and internships, there are however a few suggestions. I recommend
that the institution enlarge the sizes of the laboratories to accommodate all the students or
reduce the number of students so that every student gain well from the practicals carried out
within the laboratory. I would also recommend supply of reagents since some experiments
cannot be carried out without certain experiments.

28
REFERENCE
Azmat MA, Khan IA, Cheema HM, Rajwana IA, Khan AS, Khan AA (2012) Extraction of
DNA suitable for PCR applications from mature leaves of Mangifera indica L. J Zhejiang
Univ Sci B 13(4):239–243. Doi.10.1631/jzus.B1100194

Kim CS, Lee CH, Shin JS, Chung YS, Hyung NI (1997) A simple and rapid method for
isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nucleic
Acids Res 25(5):1085–1086

Gibson UE, Heid CA, Williams PM (2016). A novel method for real time quantitative
RTPCR. Genome Res. 6, 995–1001

A.M.Y. Jaber; N.A. Mehanna; S.M. Sultan (2009). "Determination of ammonium and organic
bound nitrogen by inductively coupled plasma emission spectroscopy". Talanta. 78: 1298–
1302. Doi:10.1016/j.talanta.2009.01.060

Manit Arya†, Iqbal S Shergill, M Williamson, L Gommersall, (2015) Basic principles of real-
time quantitative PCR. Expert Rev. Mol. Diagn.

"Molecular Breeding and Marker-Assisted Selection". International Service for the

Acquisition of Agri-Biotech Applications. ISAAA. Retrieved 2015-12-12.

29