EGERTON

UNIVERSITY

INDUSTRIAL
ATTACHMENT
REPORT
AT
KENYA
AGRICULTURAL LIVESTOCK AND RESEARCH ORGANIZATION
(KALRO) NJORO

NAME: MUMBI KINUTHIA ALEXANDER

REG.NO: S15/21469/11B
FACULTY: SCIENCE
DEPARTMENT:

BIOCHEMISTRY

BIOLOGY

COURSE: Bsc. (BIOCHEMISTRY)

SIGNATURE:________________

AND

MOLECULER

PURPOSE STATEMENT
INDUSTRIAL ATTACHMENT PRESENTED TO THE DEPARTMENT OF BIOCHEMISTRY
AND MOLECULER BIOLOGY AS A REQUIREMENT FOR THE AWARD OF THE
DEGREE IN BIOCHEMISTRY IN EGERTON UNIVERSITY.

FROM:
TO:
SUPERVISORS NAME:
MANAGER:

DECLARATION
This attachment report is my own original work and has not been presented to any examination
body. It is the accurate report of my industrial attachment at Kenya Agricultural livestock and
Research Organization.
Students Name;
Reg. no;
Sign;
Date;
Approved by;
Supervisors name;
Sign;
Date;

DEDICATION
I specially dedicate this work to The Lord God Almighty, for His grace and strength to work with
CMR-KEMRI. I would also like to extent my heartfelt gratitude to my family members; My
mum Margaret Mumbi, Uncles Moses and Duncan, and Aunt Catherine for their invaluable
financial and moral support throughout my attachment. I would also to thank my mentor Mrs.
Ruth Nditi and Mrs. Njaga for their continued moral and financial support.

Acknowledgements
I acknowledge with deep gratitude the efforts of Dr Othira Odongo a lecturer at Egerton
University towards ensuring that I get attached at KALRO Njoro. Also special thanks to KALRO
environment for providing competent technicians who tirelessly monitored our hands on skill
besides giving us reliable professional advice. The attachment coordinator at KALRO Mr.
Langat and customer care Mr. Omoso. The technicians included Miss Mary Soil lab, Mr. C.
Kimani and Mr. Manfred molecular lab (MAS lab), Mrs. Mercy (MSc) Mr. Festus and Mr. Eric
pathology lab, Mr. Odhiambo (MSc) cereal chemistry and many others. I also thank my parents
Miss Margaret and Mrs. Karana for allowing me to be on this attachment and giving me both
financial and moral support. Above all I want to thank God almighty for giving me good health
and energy. Lastly but not least, I wish to thank my fellow students who were team players and
very uplifting during the time of weakness.

TABLE OF CONTENTS
Preliminary
TITLE PAGE...................................................................................................................................1
PURPOSE STATEMENT...............................................................................................................2
DECLARATION ............................................................................................................................3
DEDICATION ...............................................................................................................................4
ACKNOWLEDGEMENT..............................................................................................................5
SECTION ONE
INTRODUCTION
ORGANIZATION PROFILE.........................................................................................................8
ORGANIZATION HISTORY........................................................................................................8
VISION...........................................................................................................................................8
MISSION .......................................................................................................................................8
OBJECTIVES OF THE CENTRE.................................................................................................9
CORE VALUES OF THE COMPANY........................................................................................10
SECTION TWO
CEREAL CHEMISTRY LAB
INTRODUCTION.....................................................................................................................11
OBJECTIVE OF CEREAL CHEM LAB...................................................................................11
HECTOLITRE............................................................................................................................11
BASIC TESTS
WHEAT AND FLOUR TESTS..................................................................................................11
MOISTURE CONTENT............................................................................................................11
FALLING NUMBER..................................................................................................................11
MILLING TEST..........................................................................................................................13
FARINOGRAGH.......................................................................................................................13
ALVEOGRAPH........................................................................................................................14
CYANIDE TEST IN CASSAVA..............................................................................................15
SECTION THREE
SOIL CHEMISTRY LAB
6

INTRODUCTION......................................................................................................................16
MATERIALS AND METHOD..................................................................................................16
TEST FOR METALS..................................................................................................................17
REAGENTS...............................................................................................................................18
STANDARD FOR NITROGEN................................................................................................19
PROCEDURE...........................................................................................................................20
METAL DETERMINATION.....................................................................................................20
SECTION FOUR
PATHOLOGY
INTRODUCTION.......................................................................................................................21
SEED HEALTH RESEARCH....................................................................................................21
SCREENING OF FUNGICIDES................................................................................................22
PLANT VIRUSES RSEARCH...................................................................................................23
METEREOLOGY........................................................................................................................24
SECTION 5
MAS LAB
INTRODUCTION...................................................................................................................26
BACKGROUND INFORMATION.........................................................................................26
MATERIALS AND METHOD................................................................................................27
RESULTS AND DISCUSSION...............................................................................................29
CONCLUSIONS........................................................................................................................32
REFERENCES...........................................................................................................................33

SECTION ONE

INTRODUCTION
Organization profile
Kenya Agricultural Research Institute Njoro (KARI NJORO) is situated 200km west of Nairobi in Njoro
district, Nakuru County along the Nakuru Mau Narok road, at an elevation of 2120 meters above sea
level.

3.2 PHYSICAL ADRESS.

Kari-Njoro,
Private bag 20107, Njoro, Kenya
Telephone: +2545161120/61528/61720
Wireless: 35140865
Fax:

+2545161576

Email: [email protected]
Website: www.kari.org, Kenya

Organization history
The history of the Centre dates back to the beginning of the century when Lord Delamare introduced
wheat in Kenya and found it difficult to grow the crop due to the problem of stem rust. This work was
based at the then Scott Laboratories, Nairobi (now called National Agricultural Research Laboratories)
from 1908. In 1927, -Njoro KARI then called National Plant Breeding Station was started at Njoro to
handle breeding work on various crops. The Centre is located in Nakuru County, 200 Km West of
Nairobi in Njoro District, and 20 Km west of Nakuru city. It lies on longitude 35 0 56' oilseed 32 South
East and on latitude 00 20' 36 North at an elevation of 2120 metres above sea level
VISION
Kari envisions a vibrant commercially oriented and competitive agricultural sector, propelled by science,
technology and innovations that respond to demands and opportunities.

MISSION
KARI-NJOROS mission is to contribute to increased productivity, commercialization and
competitiveness of wheat, oilseeds crops and other demanded agricultural products through generation
and promotion of knowledge, information and technologies that respond to clients demands and
opportunities.

OBJECTIVES OF THE CENTRE

The KARI-NJORO is guided by the following objectives in caring out its activities in process of meeting
its vision and mission to their clients.

* To generate and promote appropriate wheat, oilseed and other demanded crop varieties through
germplasm acquisition and screening, variety improvement and crop biotechnology applications for
demand driven agricultural product value chains.
* To generate and promote agronomic and pest management technologies for wheat and oil seed and
other demanded commodity agricultural product value chains.

* To develop and promote markets and marketing strategies for wheat, oil seed crops and other
demanded commodity agricultural product value chains.

* To facilitate and advocate policy options for enhancing demand driven wheat, oilseed and other
demanded commodity agricultural product value chains.

* To strengthen the capacity for implementing agricultural product value chains research through
establishing effective linkages and partnerships.

* To enhance dissemination and adoption of suitable technologies on agricultural product value chain
research within our mandate districts.
* Extension service providers to Community based organizations (CBOs, FBOs CIGs,
NGOs, etc)

CORE VALUES OF THE COMPANY

9

The institute embraces the following core values in pursuing the journey to leach and meet its mission,
vision and objectives in corporation with the clients expectations.
* Transparency, Accountability and cost effectiveness.
* Integrated and holistic approach.
* Impact performance and service delivery.
* Scientific excellence, creativity and flexibility.
* Partnership and collaborate advantage and synergies.
* Effective knowledge and information management.
* Respect of staff and clients diversity.

Guiding principles of the company

The institution is entirely guided by the following principles in meeting the clients expectation and in
providing the best professional services and advice to their clients at all times and even improving the
services to higher magnitude.

* Integrity.
* Respect.
* Justice.
* Selfness.
* Objectivity.
* Honesty.
* Leadership.
* Decency.
* Competency.
* Hard work and commitment.

10

Section Two

CEREAL CHEMISTRY LAB

Introduction to the laboratory.
Cereal chemistry is a major biochemistry arm in the organization. It is here where samples of
food crops ranging from wheat to cassava are analyzed. In the various lab sessions I was
intrigued by the scope of lessons one can gather from the institution. I was introduced into the
principle researches that occurred in the lab and they included Farinograph use, flour moisture
analyzer, baking oven, loaf volumeter, hectolitre, spectrophotometer grain analyzer, falling
number machine, cyanide test kit.

Hectolitre
Using a hectolitre

It is used measure mass in grams per 0.5 hectoliter of each strain. The results are then converted
into grams/ HL and finally into Kg/HL. The results are useful data for determining good quality
strains. Most of the strains used range from 348.5- 362.2g/ HL. It means that they are above 70
and most are god quality strain. You can always blend a strain with lower than 70 units.
While in my attachment period I acquired skills majorly done in this lab like basic tests where moisture
content, ash content, protein content, and falling number of many strains of millet were performed. The
strains I dealt with included Duma wheat strain, Eldoret strain and Njoro strain. I was able to perform
physical test that majorly dealt with flour analysis and SKCS, milling tests, dough and gluten strength
tests that involved the use of Farinograph, Alveograph machines among others. Flour starch viscosity is
another test that I learned where a rapid visco- analyzer and amylograph machines are used to determine
flour starch viscosity.

Objective of the cereal chemistry lab.

The main objective of the lab was to provide quality services majorly on cereals and other food tests
to clients. The tests included; Basic tests, Physical tests, Milling tests, Wet gluten tests and Flour starch
viscosity tests.

BASIC TESTS
Wheat and flour tests
Moisture content
During my attachment period in this lab I used a grain analyzer that auto determine the moisture
content of different wheat strains that I was working with. I was also guided on the procedures to
determine moisture content in the strains. Where moisture content is determined by heating flour
or ground wheat in an air oven and comparing the weight of the sample before and after heating.
11

Moisture content= weight f flour before heating weight of flour after heating
The amount of weight lose is the moisture content and is expressed as a percentage.
Importance of determining moisture content
Flour millers adjust the moisture in wheat to a standard level before milling
Moisture content is also an indicator of grain stability
Wheat or flour with high moisture content above 14.5% attracts molds, bacteria
and insects which cause deterioration during storage.
Other methods that I was mentioned to that are used to determine moisture content especially in
industries are ; Federal grain inspection service which uses the GAC2100 to measure moisture
content of the whole wheat.

Falling number
A falling number instrument analyzes viscosity by measuring the resistance of flour and water
paste to a falling stirrer.
I weighed a 7 gram sample of ground wheat and combined it with distilled water in a glass of
falling number tube with a stirrer and shaken it to form a slurry. Then heated the slurry in hot
water. As slurry is heated in hot water or boiling water bath at 1000c and stirred constantly, the
starch gelatinizes and forms a thick paste. The time it takes the stirrer to drop through the paste is
recorded as the falling number values. And I recorded them as follows
Wheat strains
Duma
Eldoret
Njoro

Time in seconds in first

test
352
398
286

Time in seconds
in second test
354
400
288

Average
353
399
287

Results
The falling number instrument analyses viscosity by measuring the resistance of a flour and
water paste to a falling stirrer. The results are recorded as an index of enzyme activity in a wheat
or flour sample and the results are expressed in time as seconds.

Milling tests
Buhler lab flour mill test
12

The Buhler lab flour mill test is a method followed to determine milling properties on small
wheat samples.
Procedure
Clean a sample of wheat by winnowing and hand picking of chaff and the moisture content
determined by use of the grain analyzer. Water is added to condition (temper) the wheat
overnight prior to milling.
Soft wheat requires less water and less time than hard wheat. The tempered wheat sample is run
through the mill the following day.
The mill fractions such as flour streams, bran and shorts are weighed and recorded.

Results
Wheat samples are milled too evaluate wheat milling properties including flour extraction and
the amount of non-flour components produced such as bran and shorts. The results are expressed
as the weight of flour, bran and shorts.
Farinograph
Farinograph is an instrument that I found in the cereal chemistry and food chemistry lab that is
used to determine or measure flour water absorption and dough strength.
Method

A flour sample of 50 or 300 grams on a 14% moisture basis is weighed and placed into
the corresponding Farinograph mixing bowl.
Water from a burette is added to the flour and mixed to form dough.
As the dough is mixed the Farinograph records a curve on a graph paper or filter paper
The amount of water added affects the position of the curve on the graph paper. Less
water increases dough consistency and moves the curve upward.
The curve is centered on the 500 brabender unit (BU) line +20 or -20 Bu by adding the
appropriate amount of water and is run until the curve leaves the 500-BU line.
Results

The Farinograph test measures and records the resistance of dough to mixing with paddles.
Absorption; is the amount of water required to centre the Farinograph curve on the 500-BU.
This relates to the amount of water needed for flour to be optimally processed into end products.
Absorption is expressed as a percentage.
Peak time; indicates dough development time, beginning the moment water is added until the
dough reaches maximum consistency and it gives an indication of optimum mixing time under
standardized conditions. Peak time is expressed in minutes.
13

Arrival time; this is the time when the top of the curve touches the 500-BU line. This indicates
the rate of flour hydration (the rate at which the water is taken up by the flour) is expressed in
minutes.
Departure time; is the time when the top of the curve leaves the 500-BU line. It indicates the
time when the dough is beginning to break down and is an indication of dough consistency
during processing and is expressed in minutes.
Stability time; is the difference between arrival time and departure time and it indicates the time
the dough maintains maximum consistency and is a good indication of dough strength. Stability
time is expressed in minutes.
Mixing Tolerance Index (MTI) - It is the difference in BU value at the top of the curve at peak
time and value at the top of the curve 5 minutes after the peak; it indicates the degree of
softening during mixing. Mixing tolerance index is expressed in minutes.
Weak gluten flour has a lower water absorption and shorter stability time than strong gluten
flour.

Alveograph
Alveograph determines the gluten strength of dough by measuring the force required to blow and
break a bubble of dough.
Procedure

A sample of 60 grams of flour is mixed with a salt solution to form dough.

Five 4.5 cm circular dough patties are formed and then rested in the Alveograph in a
temperature-regulated compartment at 250c for approximately 20 minutes.
Alveograph blows air into a dough partly which expands into a bubble that eventually
breaks.
Then record the pressure inside the bubble as a curve on graph paper.
Results

The results include p value, L value and W value.

Stronger dough requires more force to blow and break the bubble (higher P value)
A bigger bubble means the dough can stretch to a very thin membrane before breaking (L value)
A bigger bubble requires more force and will have a greater area under the W value.
Discussion
Processors to ensure a more consistent process and product. Weak gluten flour with low P value
(strength of gluten) and long L value (extensibility) is preferred for cakes and other
confectionery products. Strong gluten flour will have high P value and preferred for breads.

14

P value; force required to blow the bubble of dough. It is indicated by the maximum height of
the curve and is expressed in millimetres (mm).
L value; Is the extensibility of the dough before the bubble breaks. It is indicated by the length
of the curve and is expressed in millimetres (mm).
P/L ratio; Is the balance between dough strength and extensibility.
W value; Area under the curve. It is a combination of dough strength (P value) and extensibility
(L value) and is expressed in joules.
Weak gluten flour has lower p value than strong gluten flour.
Other instruments that that I was introduced to are;
Pictrate test for cyanide in cassava
Introduction
Cassava contains cyanide that is toxic and directly or indirectly causes some observed diseases of
humans in cassava growing areas. Frequent to consumption leads to inhibited iodide absorption
by the thyroid gland, a rise in plasma cyanate concentration and increased iodide excretion in
urine and thyocynate.
To reduce cassava toxicity the tubers are generally processed into different traditional foods
which may be tedious involving different steps that may require several days. Some products
require either low or medium cyanide varieties in because processing reduces the toxin to only
70% or less. Inadequate processing primary due to food shortages has led to epidemics of
cyanide related diseases e.g. spatic paraparasis (rosling 1986). Breeding is needed to develop
varieties with low or no cyanide content.

Section Three

15

SOIL
CHEMISTRY LAB
Introduction
Soil chemistry unit at KALRO Njoro
offers soil, plant, manure and water
quality analytical services. The section

SOIL
CHEMISTRY
LAB

has experienced personnel and modern

state of the art computerized
equipments to carry out the analysis.
Among the mostly evaluated nutrients
are; Soil reaction and exchangeable
acidity, Electrical conductivity in
saturated soil paste extracts,
Determination of macro nutrients such
as nitrogen, phosphorous and carbon
and Determination of micro nutrients
such as zinc, copper, magnesium,
selenium, iron and calcium
Soil reaction is one of the most
important characteristics in standard
soil analyses and has profound effect on
the availability of nutrients to plants and
on the activity of soil microorganisms.
16

Many soil chemical and biological reactions are controlled by the pH of the soil solution in equilibrium
with soil particle surfaces. (Dr.L.S. Nafuma)

Materials and methods

Preparation of soils for analysis
Air-dried, less than 2 mm
If no further instructions are given in the method for preparation of the soil, air-dried less than 2 mm
samples are analyzed.
precautions

Avoid using the motorized grinder if iron is to be determined

Avoid using brass sieves if tin or copper is to be determined. Stainless sieves are available.

procedure

Air-dry the sample on a sheet of brown paper.

Clay soils are best crushed for passing 2 mm sieve before they reach complete air-dryness.
Otherwise the crushing process is difficult.

Run the sample through a 2-mm sieve

Break up the remaining aggregates with a wooden rolling pin and again run through the sieve.
Crushing of primary sand and gravel particles is generally avoided.

The remaining gravel should be weighed and then discarded or saved for further examination.

Coarse fragment (%) = (Weight of gravel x 100)

17

Grind a complete portion of the 2-mm sieve sample to pass through the required mesh size. Use
a mortar and pestle.

Wash the mortar and pestle with distilled water followed by air drying or wiping with gauze
or filter.
TESTS FOR METALS ELEMENTS

DETERMINATION OF TOTAL N, P, Ca, K, Mg, Fe, Cu, Zn AND Mn IN SOIL AND PLANT
DIGESTS

The content of nutrient in soil is determined in the digest obtained by treating soil and plant sample with
sulphuric acid + hydrogen peroxide + selenium and salicylic acid. The principle takes into account the
possible presence of nitrates and nitrites by coupling them with salicylic acid in an acid media to form 3nitrosalicylic or 4-nitrosalicylic. These compounds are reduced to their corresponding amino acid forms
by soil organic matter. Analysis of total elements requires complete oxidation of organic matter. In this
method, concentrated sulphuric acid partially oxidizes organic matter while selenium acts as a catalyst for
the process and complete oxidation is accomplished by H2O2 additions and heating at elevated
temperatures. The main advantages of this method are that a single digestion is required (for either soil or
plant material) to bring nearly all nutrients into solution; there is no volatilization of metals. It should be
noted that various wet-ashing procedures have been proposed worldwide, depending mainly on
equipment, sample targets and personnel experience. The procedure presented here is fast, accurate and
reproducible.

REAGENTS preparation

18

Sulphuric-Selenium powder mixture: Dissolve 0.70 g of Se (1) in 200 ml of conc. H2SO4 (4). Heat to 300
OC while covering the beaker with a watch glass. The original black colour of selenium suspension turns
clear light yellow via green blue.
Digestion Mix: Dissolve 3.2 g of salicylic in 100 ml of sulphuric acid-selenium mixture (5). This mixture
should be prepared as required because it does not keep more than 48 hours.
PROCEDURE

Weigh 0.5 0.001 g of oven-dried (70 OC), finely ground plant tissue or soil passed through
a 0.25-mm (60-mesh) sieve into labeled, dry and clean digestion tubes. (Note: Include
standards[see below for respective elements ])

Add 4 ml of the digestion mix to each tube and the reagent blanks for each batch of samples.

Digest at 110 OC for 1 hour.

Remove, cool and add three successive 1-ml portions of hydrogen peroxide (H2O2) and
replace on the block digester.

Raise the temperature to 330 OC and digest for 2 hours. The solution should be colorless with
only silica at the base of the tubes. If the solution is coloured continue digesting until the
solution is colorless.

Remove tubes and after cooling add sufficient DD water to bring the solution to the 100-ml
mark.

Mix thoroughly and allow solution to stand overnight so that the silica settles out and then
decant clear solution into storage tubes (supernatant of digest)

19

STANDARDS FOR NITROGEN

Stock Standard Solution (1000 ppm N): In a 1-L volumetric flask, dissolve 4.717 g of ammonium
sulphate [(NH4)2SO4] that has been dried for 2 hours at 110 OC, in about 800 ml of DD water. Dilute to
the mark and invert three times
Working Standard Solution (20 ppm): In a 1-L volumetric flask, dilute 20 ml of stock standard solution
(1., above) to the mark with DD water. Invert three times
Working Standard Series (set of six): To six 100-ml digestion tubes add, respectively, 0, 2, 5, 10, 15 and
20 ml of the working standard solution (2., above). Add DD water to each tube to bring the volume to
approximately 20 ml. Add 4 ml of digestion mix and mix thoroughly. Then digest these solutions just like
the samples. The standard series contains 0, 2, 5, 10, 15 and 20 ppm NH4-N.

PROCEDURE
[1] Wash plant parts in distilled water containing 0.1 - 0.3% detergent (phosphate-free if P analysis is
required). Plant tissue should be placed in container with the wash solution and should be gently moved
within the container.
[2] Rinse in distilled water
[3] Place in paper bags and dry immediately. Plant samples are dried for 24 to 36 hours in a convection
oven set at 70 O C.
[4] After drying grind the samples in a Wiley Mill (If analysis of Fe is not required) or using mortar and
pestle (if Fe is important) to eliminate sampling errors when the sample is weighed. Plant tissue should be
20

finely ground to achieve this objective. Generally, plant tissue is ground to pass a 1-mm sieve. Before
weighing milled plant tissue for chemical analysis, the oven-dried plant samples should be placed in a
convection oven set at 70 O C overnight and then allowed to cool in desiccators.
DETERMINATION OF PHOSPHORUS, POTASSIUM AND NITROGEN BY SODIUM
BICARBONATE METHOD OF EXTRACTION
PRINCIPLES
Phosphorus is extracted from the soil with 0.5M NaHCO3 at a nearly constant pH of 8.5. In calcareous,
alkaline or neutral soils containing Ca phosphates, this extract decreases the concentration of Ca in
solution by causing precipitation of Ca as CaCO3; as a result, the concentration of P in solution increases.
In acid soils containing Al and Fe phosphates such as variscite and strengite, P concentration in solution
increases as the pH rises. Secondary precipitation reactions in acid and calcareous soils are reduced to a
minimum because the concentrations of Al, Ca, and Fe remain at low level in 0.5M NaHCO3

Section Four

PATHOLOGY
Introduction
This section deals with crop diseases and their control measures. The main crops that
are dealt with are: (I) Small seed cereal crop like- wheat, barley, triticale etc.
(ii) Oil crops e.g. sunflower, rapeseed, and soybean
(iii) Other crops- other crops in mandate region
This is through the General Advisory Services (GAS)
The section has eased its work by creating the following divisions: 21

(i) Seed health research

(ii) Screening of fungicides
(iii) Plant virus research-BYDV (Barley Yellow Dwarf Virus)
- TSWV (Tomato Spotted Wilt Virus)
-BSV (Banana streak virus)
(iv) Meteorology
The center received a new automated meteorological monitoring unit from Geneva
Switzerland.
The above subsections are run by a research officer assisted by the technical assistance
and other members of the section
Seed Health Research
This subsection works hand in hand with the breeders of wheat and oil crops from the
center and outside from farmers. The concern is mostly on the seed. After getting the
required seed, various processes are undergone and analyzed. The processes differ from
cereals and oil crops. For cereals e.g. wheat, they include: : - Giving the wheat accession numbers, which include the entry, variety and place
collected.
:
: - On the seventh day the microscope work is done to find out whether the pathogen is
either on the seed in the seeds or with the seed. This involves systematic placing the
grains on a filter paper in a systematic circular motion. They are the store for up to 4
days before observation under a microscope.
Table 4.1 Some of the fungal pathogens recorded on wheat seeds in Kenya are:
Pathogen_
Phoma ssp
Bipolaris sorokiniana
Fuserium semitectum_
Graminearum
Fusarium solani
Fusarium equiseti

% Infection
96.0
51.0
45.0
27.0
3.0
9.0

For oil crops, the main interest is on soybeans. The process followed includes: : - Sampling the soybean and picking randomly. Count 100 seeds and plate 10 seeds per
Petri dish with blotter paper dipped in distilled water.
: - On the seventh day, microscopic observation is carried out to find out whether the
pathogen is either on the seed, in the seed or with the seed.
Some of the fungal pathogens recorded on soybeans seeds in Kenya include: (i) Phoma spp
(ii) Phomopsis sp.
(iii) Fusarium moniliforme
(iv) Colletotricum dematium
22

(v) Cercospora kikuchi

Screening of fungicides
Table 4.2 A Flow chart on the procedure for screening fungicides.
Agro-chemical company
_Pesticide control board (Nairobi)
_Station (National Plant Breeding Center)
_Section (Pathology) screen for 3 years
1st and 2nd year- Give tentative recommendation.
Usually the screening program I take 3 years. Some of the diseases controlled by the
use of fungicides include: Table 4.3 Common diseases of wheat analyzed
Disease
Symptoms
Stripe
Yellow pustules in parallel
rust(Puccinastriformis)
lines on the leaves -Yellow
spores inside glumes
Stem rust (Puccina graminis Elongated reddish brown
tritici
pustules on stem, leaf and
sheath
Loose smut_
Powdery black spores
Tan spot
Take all

Irregular spots on leaves

Tan in colour with brown
spots near the center
Bleached white heads
Ripe prematurely Plant
easily pulled from soil

Control
Planate 1.5l/haTilt 0.5 l/ha , Resistant varieties
Resistance varieties Plant at
least three cultivars
Use certified seed Dress with
vitavax 200 ff and Bayton
Crop rotation with soybean,
potatoes or sunflower
Crop rotation with cereal crops

Table.13: Common diseases of soybeans analyzed.

Disease
Anthracnose

Soybean rust

Symptoms
Elongated spots on the
upper surface Infected pods
have brown spots with rust
boarders
-Seeds have
lesions of various sizes
covered with small fruiting
bodies
Brown or reddish brown
23

Control
disease free seed
-crop rotation
-seed treatment
-spray with Benlate

resistant cultivars -use of

_Pod and stem blight

Damping off

pustules on the affected

parts
cause latent infection
throughout the plant
Seeds are cracked,
shrivelled and no
germination
Seeds do not germinate,
soft marshy brown and
disintegrate

protectant fungicides
_-resistant cultivars
-rotation with cereals
seed treatment fungicides
seed treatment
-foliar sprays

Advisory services
After carrying out research on seed health and identified the pathogen causing a disease,
the research officers advice to the farmers and the breeders on the type of control to on
the crops.
Plant virus research
This research is under a research officer as a virologist. Deals mainly on B.Y.D.V. and
T.S.W.V
Plant Virus Detection
1) Diagnostics of plant viruses:
Indefinite: -Symptoms check-up e.g. yellowing of leaves
-Host plants/test plants
Definite: - Serological tests
(i) Precipitation Test
This test needs dilution of about 10-3 of virus. Antigen in a tube plus antibody in a tube
then mixed. If precipitation occurs then the test is correct, no precipitation shows no
antibodies.
(ii) Gel Diffusion
The test needs dilution of about 10-5 whereby uses of an agar jell is used. Two holes are
made whereby in one hole an antibody is placed and in the other hole an antigen. The
antibody and antigen will start diffusing out and will come to a point where they will
meet forming a precipitation.
(iii) Elisa-Test (Enzyme-linked Immunosorbent Assay for the Detection of plant
virus)
This is the mostly and currently used in the section. It needs a dilution of 10-3 hence
used to detect small quantities of viruses.
Direct Elisa is carried out and follows the following procedure: : - Have a well done on immunological plates made from special material whereby the
antibodies and antigens are able to coat.
: - Coat the well with antibody and incubate for some time to ensure uniform coating
24

: - Coat well with antigen, which is a virus particle, extracted from the plant sap.
: -Detect where combination occurs by using the same antibody to detect the antigen.
: -To detect whether the antigen has the virus, use the enzyme label which is mostly
alkaline phosphates because it is able to change the colour very first when there is the
combination on specific proteins. In this case antigen and antibody hence the title Elisa
(enzyme linked)
METEOROLOGY
This is a science of weather, which started in 1873 in Italy and to Geneva in 1950. The
type of weather station in the center is meteorological which has the following
equipment: -Computerized weather Station.
:
:
:
:
:
:
:
:

-Anemometer for measuring wind speed/velocity

-Wind vane for wind direction
-Standard rain gauge for rainfall
-Radiometer for radiation
-Maximum and minimum thermometers for temperatures
-Barometer for pressure
-Stephenson screen, which is painted white
-Radio calls for sending information to Dagoreti

Importance of meteorology
1. Agronomists are able to know the direction of wind and when to spray their
chemicals.
2. Pathologists are able to know the inoculation time.
3. Entomologists are able to know the direction from which the insects are coming
from.
4. Cereal breeders and farm are able to know the amount of rainfall.
5. for checking on the moisture in the soil, the lower the temperature, the higher the
moisture .

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Section Five

MARKERS ASSISTED SELECTION (M.A.S) LAB

Introduction
Started in 1994, the markers assisted selection lab is Located on the 2nd floor middle block of the
KALRO lab complex. It analyses both plant and bacterial molecular markers and to some extent
isoenzymes.
Molecular markers are inherited DNA sequences that can be tracked. The use of these markers is
based on naturally occurring DNA polymorphism. Thus a marker must be polymorphic i.e. it
must exist in different forms so that chromosome carrying the mutant gene can be distinguished
from the chromosome with the normal gene by a marker it carries. Genetic polymorphism is the
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simultaneous occurrence of a trait in the same population of two or more discontinuous variants
or genotypes
When used together with electrophoretic techniques, molecular markers can determine DNA
quality and quantity. This will further be discussed using sample protocols.

Background information
MARKER
RFLP

ADVANTAGES
Unlimited number of loci

DISADVANTAGES
Labor intensive

Co dominant

Fairly expensive

Many detection systems

Large quantity of DNA needed

Can be converted to SCARs

Often very low levels of polymorphism

Robust in usage

Can be slow (often long exposure

Good use of probes from other

species

times)
Needs considerable degree of skill

Detects in related genomes

RAPD

No sequence information required

Results obtained quickly

Highly sensitive to laboratory changes

Fairly cheap

Low reproducibility within and between

No sequence information required

Relatively small DNA quantities required

laboratories

Cannot be used across populations nor across

species

High genomic abundance

Often see multiple loci

Good polymorphism

Dominant

Can be automated
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SSR

High developmental and startup costs

Species-specific

Sometimes difficult interpretation

because of stuttering

Usually single loci even in polyploids

Materials and methods

a) Genomic DNA Extraction using Charles CTAB protocol

Pre heat 500l CTAB in 65C water bath.

Place 0.2 to 0.5g plant material into 2.0ml centrifuge tube and place into liquid nitrogen
(some of the liquid nitrogen can get into the tube), grind to a powder.

Add plant material to the pre-heated CTAB and add1%-mercaptoethanol (5l) to the
centrifuge tube and incubate at 65C for 1hr. Vortex well at least every 20min.

Centrifuge for 8min. at 13,000rpm, transfer top layer to new micro centrifuge tube.

Add 240l phenol/EB pH 8.0 and 260l 96:4, vortex well, centrifuge for 3min., transfer
top layer to a new micro centrifuge tube.

Add 500l 96:4, vortex well, centrifuge for 3min transfer top layer to a new micro
centrifuge tube.

Add 100l 10M NH3 OAc and 1000l 100%EtOH, vortex, incubate in ice bath for
10min. Centrifuge for 10min. at 13,000 and discard supernatant. Be careful not to suck
up the pellet.

Wash pellet in 500l cold 70% EtOH, invert 5X; incubate 10min. in an ice bath.

Centrifuge for 2min. to secure the pellet, discard the supernatant.

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Dry the pellet. (Centri-vac 2min.)

Add 50l EB Buffer, store in 4C overnight before quantifying.

CTAB Solution:
2g CTAB2g PVP,28ml 5M NaCl (29.22g/100ml),4ml EDTA pH 8.0,10ml 100mM TrisHCl
pH8.0,1% -mercaptoethanol (just before use)Prepare the solution without CTAB, PVP, mercaptoethanol and autoclave for 20 min: After autoclave add CTAB and PVP; heat to dissolve
CTAB and PVP on the hot tray. Add the 2% -mercaptoethanol directly to the extraction vial
(10l/500l of CTAB solution). Pre-heat the solution in a 65C water bath before use.
b) DNA extraction protocol( Dellaporta et al 1983, modified by MARI)

Results and discussions

General Considerations and Rationale on DNA extraction
Three processes are vital for successful nucleic acid extraction. they include:
1) Inhibition of nucleases,
2) Removal of proteins,
3) Physical separation of the nucleic acid from other cellular components.
NB. Nuclease inhibition and removal of proteins are not mutually exclusive and often are
accomplished in a single step (such as phenol extraction).

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Inhibition of Nucleases
Detergents inhibit nucleases and help separate the proteins from the nucleic acids. A common
detergent is sodium dodecylsulfate (SDS). Avoid the use of potassium salts or temperatures
below 10 C with SDS since this may cause precipitation of the detergent. One of the most
widely used detergents for plant DNA extraction buffers is CTAB (cetyltrimethyl-ammonium
bromide). CTAB is most often used in a 2% (w/v) solution (Rogers & Bendich 1985, Doyle &
Doyle 1987, Nickrent 1994).
Removal of Proteins
Phenol is a strong denaturing agent for proteins. In phenol extractions, proteins partition into the
organic phase (and interface) whereas nucleic acids partition in the aqueous phase. Usually
phenol is used in a 1: 1 mixture with chloroform since deproteinization is more effective when
two different organic solvents are used simultaneously. In addition to denaturing proteins,
chloroform is useful in removing lipids and a final chloroform extraction helps to remove the last
traces of phenol. The isoamyl alcohol helps with the phase separation, decreases the amount of
material found at the aqueous and organic interface, and helps reduce foaming.
Antioxidants such as 8-hydroxyquinoline or -mercaptoethanol are often added to phenol.
During phenol extractions, the pH of the buffer is important in determining whether DNA and/or
RNA are recovered. At pH 5-6, DNA is selectively retained in the organic phase leaving RNA in
the aqueous phase (hence water saturated phenol is useful for RNA extractions). At pH 8.0 or
higher, both DNA and RNA are retained in the aqueous phase. Phenol can be stored under buffer
for up to one month.

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Protease
The most common proteolytic enzyme used in nucleic acid extractions is protease K (e.g. Sigma
P4755) which is generally made up in 50 units/ml aliquots and stored frozen for immediate use
during extractions. Solutions are generally incubated with this enzyme at 37 C for at least one
hour.
Other Extraction Buffer Components
Two common reducing agents found in extraction buffers are -mercaptoethanol or dithiothreitol
(DTT). EDTA is also present to chelate Mg+2 ions thus mediating aggregation of nucleic acids to
each other and to proteins.

Ethanol and/or Isopropanol Precipitation

The most common method of concentrating nucleic acids is with alcohol precipitation. This
occurs in the presence of a salt (see below) at low temperatures (-20 C or less). Either 2.0
volumes of ethanol can be used to precipitate DNA or 0.6 volumes of isopropanol. Isopropanol is
often used for the first precipitation, but not for final ones because it tends to bring down salts
more readily than ethanol. Isopropanol can also be used when the volume of the tube will not
allow the addition of 2 volumes of ethanol. The nucleic acid is collected by centrifugation at
10,000 rpm for ca. 20 - 30 minutes.
The most common monovalent cations used in nucleic acid precipitations are shown in the
following table:
* Filter sterilize, do not autoclave

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The choice of salt is determined by the nature of the sample and the intended use of the nucleic
acid. Samples with phosphate of greater than 10 mM EDTA should not be ethanol precipitated
because the salts will come down with the nucleic acid.

CONCLUSION
The attachment was of great importance to us the learners since we were exposed to different
areas of science and also to put in practice what we learnt theoretically in the university. What
fascinated was specifically in the Molecular laboratory where we were able to develop, extract
and view DNA from different plant samples. However, the other laboratories in the organization
were equally of great help to the upcoming scientists. Given another chance I would definitely
without hesitance want to work at the organization.

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REFERENCES
1. Lubert Strayer (1998) Biochemistry, W.H Freeman and Company, New York.
2. "Bacteria." Biology Online. 22 July 2008. 2 Dec. 2008 <http:// www.biologyonline.org/dictionary/bacteria>.
3. Madigan, Michael, John Martinko, Paul Dunlap, and David Clark. Brock Biology of
Microorganisms. 12th ed. Boston: Benjamin-Cummings Company, 2008. 414-15.
4. Potter, Dr. Beth. "Biochemical Tests." Microbiology 202. Penn State Erie, 30 Oct. 2008
11 Nov. 2008.

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