A REPORT ON STUDENT INDUSTRIAL WORK

EXPERIENCE SCHEME (SIWES)

UNDERTAKEN AT

NATIONAL CENTRE FOR GENETIC RESOURCES AND BIOTECHNOLOGY

(NACGRAB) APATA, IBADAN, OYO STATE.

PERIOD OF ATTACHMENT: April 06, 2021 – September 22, 2021

By

IHEJIRIKA, CHRISTIANA C.
U18/NAS/BTG/076

SUBMITTED TO

THE SIWES COORDINATOR,

DEPARTMENT OF BIOLOGICAL SCIENCES,

FACULTY OF NATURAL SCIENCE AND ENVIRONMENTAL STUDIES,

GODFREY OKOYE UNIVERSITY, ENUGU STATE.

IN PARTIAL FULFILMENT FOR THE AWARD OF BACHELOR OF SCIENCE (B.SC) DEGREE IN

BIOTECHNOLOGY.
 ACKNOWLEDGEMENT
I acknowledge with unalloyed gratitude, my parents; Mr and Mrs Appolos Ihejirika, my HOD in
person of Dr. Felix Onyia and all lecturers of biological science department, the Director of
National Center for Genetic Resources And Biotechnology in person of Dr. S.E. Aladele, all staff
of National Center for Genetic Resources And Biotechnology, My SIWES coordinator, Mr Hassan
O and others for their contributions to my academic pursuit. I pray that almighty God will grant
them their heart desires.
However, I wish to acknowledge the special effort of my Industrial based supervisor who work
tirelessly in the perfection of this report and every other individuals who has contributed in one
way or the other to the success of my SIWES internship.
 ABSTRACT
The Students Industrial Work Experience Scheme (SIWES) is a training designed and put
forward to expose, prepare and familiarize the students with different methods and experience
needed for them to carry out jobs relating to their field of study effectively and efficiently. The
training is also designed to expose students to the practical aspect of their course of study,
what they could not be taught in school as well as the needed experience in handling
equipment, machinery and skills that are provided and those that cannot be provided by their
educational institutions but are available in the organization in which they were attached. This
report has been prepared and divided into 4 chapters for the purpose of making it clear to the
readers. Chapter one of this report shows the introduction and objective of the report SIWES.
The chapter two comprises of the organization of industrial attachment, brief history and
location in which the SIWES has been carried out, objectives, market services situation of
NACGRAB, the industries research mandate, brief discussion on the economic environment in
which NACGRAB operates, management structure, various units in the industry and their
functions. The chapter three shows training acquired and achievement in the following units in
which student was posted; molecular biology laboratory, tissue culture laboratory and seed
gene bank laboratory. This chapter covered all the techniques and practical student was
exposed to during the period if industrial training. The chapter four comprises of conclusion,
experience gained, challenges encountered and recommendation.
 TABLE OF CONTENT
CHAPTER SUBJECT PAGE
TITLE PAGE i
ACKNOWLEDGE ii
ABSTRACT iii
TABLE OF CONTENTS iv
LIST OF TABLE v
LIST OF FIGURES vi
1 INTRODUCTION AND OBJECTIVE OF REPORT (SIWES) 1
1.1 HISTORICAL BACKROUND OF SIWES 1
1.2 GENERAL OBJECTIVE OF REPORT 2
2 ORGANIZATION OF INDUSTRIAL ATTACHMENT 5
2.1 BRIEF HISTORY AND LOCATION OF NACGRAB 6
2.2 OBJECTIVES OF NACGRAB 7
2.3 BRIEF WRITE UP ON THE INDUSTRY IN NIGERIA / MARKET SERVICES SITUATION 8
2.4 NACGRAB RESEARCH MANDATE 9
2.5 BRIEF DISCUSSION ON THE ECONOMIC ENVIRONMENT IN WHICH NACGRAB 10
OPERATES.
2.6 MANAGEMENT STRUCTURE OF NACGRAB 11
2.7 VARIOUS UNITS IN NACGRAB AND THEIR FUNCTIONS 12
3 TRAINING ACQUIRED AND ACHIEVEMENT 13
3.1 MOLECULAR BIOLOGY LABORATORY 13
3.1.1 INSTRUMENTATION IN THE MOLECULAR BIOLOGY LABORATORY 14-20
3.1.2 EXPERIENCE GAINED IN MOLECULAR BIOLOGY LABORATORY / TECHNIQUES 21-30
USED IN MOLECULAR BIOLOGY LABORATORY
3.2 TISSUE CULTURE LABORATORY 31-33
3.2.1 TECHNIQUES USED IN PLANT TISSUE CULTURE 34-40
3.3 SEED GENE BANK LABORATORY 41
3.3.1 ACTIVITIES CARRIED OUT AT SEED GENE BANK UNIT 41-44
4 CONCLUSION 45
4.1 SKILLS DEVELOPED/EXPERIENCE GAINED 46
4.2 CHALLENGES ENCOUNTERED DURING INDUSTRIAL TRAINING 47
4.3 RECOMMENDATION 48
 LIST OF TABLE
No NAME OF THE TABLE PAGE NO

1 Materials used in plant tissue culture 33

LIST OF FIGURES
NO NAME OF THE FIGURE PAGE NO
1 Organizational chart of NACGRAB 11
2 Non refrigerated centrifuge 14
3 Vortex mixer 14
4 Magnetic stirrer 15
5 pH meter 15
6 Electrophoresis chamber 15
7 Orbital shaker 16
8 Refrigerated centrifuge 16
9 Weighing balance 16
10 Nanodrop Spectrophotometer 17
11 PCR machine 17
12 Gel documentation unit 18
13 Incubator shaker 18
14 Micropipette 18
15 Microwave 19
16 Fume chamber 19
17 -40°c freezer 19
18 Water bath 20
19 Result of gel electrophoresis 30
20 Acclimatization chamber 40
21 Screen/green house 40
22 Germplasm flow chart 41

CHAPTER ONE
 INTRODUCTION
1.1 HISTORICAL BACKROUND OF SIWES
The student Industrial Work Experience Scheme (SIWES) is the accepted skills training
programme, which forms part of the approved minimum academic standards in the various
degree programme for all Nigerian Universities, established by ITF (Industrial Training Funds) in
the year 1973 to solve the problem of lack of adequate proper skills for employment of tertiary
institution graduates by Nigerian Industries. It is an effort to bridge the gap existing between
theory and practice of engineering and technology, science, agriculture and other professional
educational programme in the Nigerian tertiary institutions. It is aimed at exposing student to
machine and equipment, professional work methods and ways of safe-guarding the work areas
and the workers in industries and other organizations. The minimum duration for the SIWES
should normally be 24 weeks except for engineering and technology programme where the
minimum duration is 40 weeks; the scheme is a tripartite programme, involving the students,
the Universities and the Industries (employers of labour). This scheme is funded by the Federal
Government of Nigeria and jointly coordinated by the Industrial Training Fund (ITF) and the
National Universities Commission (NUC).
It is on this note that I, Ihejirika Christiana, participated in the programme which commenced
April 6th to September 22nd, at National Center for Genetic Resources and Biotechnology
(NACGRAB), Apata, Ibadan, Nigeria.

1.2 GENERAL OBJECTIVES OF REPORT

The major aim and objective of the Student Industrial Work Experience Scheme (SIWES) report
is to give a brief description of work experience(s) gained. This report also covers the major
aims and objectives of the Student Industrial Work Experience Scheme (SIWES) which are:
1. To expose the student to industrial environments and enable them to develop
occupational competencies for them to contribute their own quota to the national
economy and scientific development after graduation.
2. To allow the student gain the technical experience on how to behave and relate with
people in working environment.
3. The scheme expose student to industry based skills necessary for a smooth transition
from the class room to the world of work.
4. It affords student of tertiary institutions the opportunity of being familiarized and
exposed to the needed experience in handling machinery and equipment which are
usually not available in the educational institutions.

1.3 BODIES INVOLVED IN THE MANAGEMENT OF SIWES

There are five bodies that are involved in the operation of SIWES in Nigeria. They include the
following;

 The Federal Government

 The Industrial Training Fund (ITF)
 The Collaborating Agencies (NUC, NBTE and NCEE)
 The Institution
 The Employer

1.3.1 THE FEDERAL GOVERNMENT

The Federal government is charged with the following responsibilities;

 Make fund available to the Federal Ministries of Industries to fund the scheme.
 Make it mandatory for all establishments to offer places to the student for attachment.

1.3.2 THE INDUSTRIAL TRAINING UNIT (ITU)

 To organize workshops, seminars and conferences on SIWES.

 To compile the list of employers and available places for industrial experience and
forward such lists to the coordinating agencies.
 To provide the logistic materials needed for the administration of the scheme.

1.3.3 THE NIGERIAN UNIVERSITIES COMMISSION (NUC)

 To establish SIWES coordinating agencies.

 To appoint full time industrial coordinators to operate the scheme at the agency level.
 To approve SIWES master and placement lists and forward such lists to ITF.
 To involve the minimum National aid program for supervised Industrial activities for
approved SIWES courses.

1.3.4 THE UNIVERSITY

 To establish SIWES coordinating units and appoint departmental and faculty

coordinators within the institution.
 To assess the students’ performance and award grade accordingly.
 To help secure placement of students on attachment with employers.
  To help in submitting the ITF forms to the training fund at the end of the program.
 To organize orientation programs for students to prepare them for industrial training.

1.3.5 THE EMPLOYER

 To accept students for Industrial Attachment as stipulated in ITF Decree No. 47 as

amended (1990).
 To participate fully in the assessment of students by completing the necessary
instruments e.g. ITF form 8, logbook etc.
 To allow students have access to their facilities.
 To appoint an Industry-based Supervisor for students on attachment
 CHAPTER TWO

ORGANIZATION OF INDUSTRIAL ATTACHMENT (NACGRAB)

2.1 BRIEF HISTORY OF INDUSTRY AND LOCATION

National Centre for Genetic Resources and Biotechnology (NACGRAB) was established in 1987 by the
Federal Ministry of Science and Technology (FMS&T) in other to conduct research, gather data and
disseminate technological information on matters relating to genetic resources conservation, utilization
and biotechnology applications. The Centre, backed by Decree 33 of 1987 regulates the seed, livestock
and fisheries industries through its Varietal Release Committees.

In January, 2004 the Center was up-graded to the status of a parastatal. However, during the
reorganization of parastatal and agencies under the Federal Ministry of Science and Technology in 2007,
NACGRAB was merged with National Biotechnology Development Agency (NABDA), NACGRAB through
its various programme, has continued to contribute significantly to the conservation of the rich genetic
resources of the nation.

The National Centre for Genetic Resources and Biotechnology is located at NCRI, Moor
Plantation, Ibadan/Abeokuta road, Apata, Ibadan, Oyo State.
2.2 OBJECTIVES OF NACGRAB

 Conservation, preservation and maintenance of valuable plant and animal genetic resources, for
immediate utilization and posterity;
 Development of animal genetic resources (poultry, snailry, fisheries etc).
 Application of tissue culture for plant conservation and overall agricultural development;
 Servicing of the activities of the National Committee on Naming, Registration and Release of
Crops Varieties, Livestock Breed and Fisheries;
 Networking and coordinating activities in the development of capacities in plant and animal
genetic resources.

VISION

A Centre that is committed to the conservation of the rich Genetic Resources of the nation, with a view
to enhancing agricultural, economic and social development.

MISSION

Nigeria is endowed with diverse biological heritage, which needs to be maintained for the purpose of
utilization and for posterity. NACGRAB is to ensure the conservation and sustainable use of these rich
biodiversity through Research and Development.
2.3 BRIEF WRITE UP ON THE INDUSTRY IN NIGERIA / MARKET SERVICES SITUATION

National Center for Genetic Resources and Biotechnology, Moor Plantation, Ibadan is grouped under
Research Institutions for the development of Science and technology with a view to Forster Agricultural
sector and National Food Security through several measures of conservation of valuable genetic
resources for sustainable utilization. NACGRAB is established to conduct research to develop facilities
for the identification of national research need and priorities and to regulate the utilization of genetic
resources, genetic engineering and biotechnology and for the training of technical and sub-technical
personnel for the service in the company. NACGRAB is a core research and educational institution and
was established in 1987 by the Federal Ministry of Science and Technology (FMS&T) to conduct
research, gather data and disseminate technological. And services rendered by NACGRAB are strictly
Non-profitable; rather for study, conservation and improvement of practices.
2.4 NACGRAB RESEARCH MANDATE

National Center for Genetic Resources and Biotechnology in Nigeria market renders the following
services via its mandate;

 Exploration, collection, identification, evaluation, characterization, storage and conservation of

rich stock of both animal and plant germplasm material.
 National coordination of genetic resources programme and its sustainable utilization.
 Fostering relationship with other national satellites genetic research centres located in research
institutes, universities and polytechnics as well as the international organizations and centres of
programmes concerning genetic resources and biotechnology application.
 Arrest rapid erosion and loss in the country’s crop and animal genetic resources caused by
cultivation, urbanization rural development, desertification, national catastrophes etc.

 Documentation appropriately the germplasm stocks held by the center, research institutes and
relevant organizations.
2.5 BRIEF DISCUSSION ON THE ECONOMIC ENVIRONMENT IN WHICH NACGRAB OPERATES.

NACGRAB'S programmes cut across several sectors under Nigeria economic environment ranging from
Science and Technology Development down to Agricultural production and Food Security. To execute
the mandate effectively and ensure proper running of activities within the organization, NACGRAB
collaborate with indigenous and international research institute, universities and biotechnological
companies abroad (Ingaba biotechnology] considering the location of NACGRAB, it is not the only
research in the area. Other research institute that are few kilometers away include: National institute of
horticultural research [NIHORT] institute of agricultural research and training [IAR & T] liaison with other
relevant international organization e.g. international plant genetic research institute [IPGRI], food and
agricultural organization [FAO] and the international institute of tropical agriculture [IITA].
2.6 MANAGEMENT STRUCTURE OF NATIONAL CENTER FOR GENETIC RESOURCES AND
BIOTECHNOLOGY.

NACGRAB is headed by a Director/Chief Executive Officer (CEO). He is responsible for daily operations at
the Center. He also oversee the activities of various units at the Center.

Fig 1: Organizational chart of NACGRAB

2.7. Various units in NACGRAB and their functions

The Center has a total of seven (7) units namely: Plant Genetic Resources (PGR), Animal Genetic
Resources (AGR), Biotechnology, Administration, Accounts, Information and Documentation, each unit
has a head who oversees/coordinates the operations of the unit and in turn reports directly to the
Director/CEO.

Animal genetic resources

AGR unit of NACGRAB is one of the core Units of the Centre established in the year 2004 and currently
has the following sections: poultry, fishery, snailry, small ruminants, cane-rat and rabbitry.

The functions of the Unit include the following:

- Exploration, collection and conservation of various indigenous animal genetic resources

- Preservation of endangered animal genetic resources

- Genetic characterization and evaluation of animal genetic resources in Nigeria

- Conduct research in conservation and utilization of animal genetic resources including the use
biotechnology tools

- Assist National Varietal Release Committee (NVRC) in the Livestock breeds and Fisheries strains
Naming, Registration and Release Activities.

Biotechnology unit

Biotechnology harnesses cellular and bio-molecular process to develop solution that helped solve
problems in many areas including agriculture and environment. The biotech unit of the centre was
established in 1999 to handle issues relating to research and development of genetic resources, in other
to complement the conservation mandate of the Centre using simple biotechnology tool.

The unit is divided into sub units: Plant tissue culture section and Molecular biology section.

Tissue culture: This section mostly supports the mandate of the Centre. Tissue culture unit engaged
mainly in in-vitro conservation, regeneration and multiplication of both agronomic and tree crop
species, on a nutritional medium under an aseptic environmental condition. The Tissue culture
laboratory housed some certain important sections which are: Growth room/Transfer section, Media
preparatory room, washing and storage room, screen house, post flask and the TIBS (Temporary
Immersion Bioreactor System).

Molecular Biology Laboratory: This section helps in characterization and evaluation of plant and animal
genetic resources in the country. Also molecular characterization and fingerprinting of germplasm
collections using molecular makers approach for identification, reduction of vast collections to their
genetic variants and construction of fine phylogenetic trees etc.
 Chapter 3
TRAINING ACQUIRED AND ACHIEVEMENT

3.1 MOLECULAR BIOLOGY LABORATORY

The molecular biology laboratory is a section of NACGRAB responsible for the characterization
and evaluation of plant and animal genetic resources.
The activities carried out by the molecular biology lab includes
i. Molecular fingerprinting of collected and conserved germplasm using molecular markers
approach for the purpose of identification and reduction of vast collections to their genetic
variants.
ii. Molecular screening of genetic resources for traits of economic and medicinal importance.
iii. Pathological screening and molecular diagnosis of germplasm infections from plants, animals
and microbes.
3.1.1 INSTRUMENTATION IN THE MOLECULAR BIOLOGY LAB
The instruments/equipment used in the molecular lab to carry out analysis are as follows;

1. Non- refrigerated centrifuge: An equipment used to separate liquids based on

density. It is used in the lab during DNA isolation and purification. It makes use of
micro centrifuge tubes.

Fig 2: Nọn-refrigerated centrifuge

2. Vortex mixer: it is used to mix samples in small tubes rapid

Fig 3: vortex mixer

3. Magnetic Stirrer: a device that employs a rotating magnetic field to cause a stir bar
immersed in a liquid to spin very quickly, thus stirring it. Used in reagent and media
preparation.
 Fig 4: magnetic stirrer
4. pH meter: an electronic device used to measure the acidity or alkalinity of an aqueous
solution.

Fig 5:pH meter

5. Electrophoresis chamber: It consists of electrophoresis power pack, the gel tank, tray
and the gel comb. These are used for agarose gel electrophoresis to separate DNA
molecules based on their size and charges whereby DNA migrates from the negative
pole to the positive pole. The tank is filled with buffer and the power pack supplies
power to it.

Fig 6: Electrophoresis chamber

6. Orbital shaker: It is used to vigorously shake or agitate samples for easy lysing of the
sample.
 Fig 7: orbital shaker

7. Refrigerated centrifuge: It is used for spinning samples that need consistent range of
temperature.

Fig 8: refrigerated centrifuge

8. Weighing balance: It is used to weigh samples in gram.

Fig 9: weighing balance

9. Nanodrop spectrophotometer: An instrument used to quickly and easily quantity and

access the purity of samples such as DNA, RNA, and proteins.
 Fig 10: Nanodrop spectrophotometer

10. Thermal cycler/ Thermocycler/PCR machine: it is a device that replicates DNA

fragments into copies through a process of alternating heating and cooling of the
machine.

Fig 11: PCR machine

11. UV documentation unit: Used for the imaging and documentation of nucleic acids
(e.g. DNA) suspended within polyacrylamide/agarose gel.

Fig 12: UV documentation unit

12. Incubator shaker: It is used for incubation and for the growing of inoculum. It is used
in genotyping, somatic embryogenesis, viral indexing and genetic transformation.

Fig 13: Incubator shaker

13. Micropipette: Used to accurately and precisely transfer volumes of liquid in the
microliter range.

Fig 14: Micropipette

14. Microwave: It is used for quick melting of agarose, during gel electrophoresis

Fig 15: Microwave

15. Fume chamber/cupboard: It is a chamber where toxic and chocking reagents are kept
to reduce the impact of the chocking gas by producing sterile air and then suck out
polluted air.

Fig 16: Fume chamber

16. Low temperature freezer (-40°C): This acts as a DNA bank where extracted DNA or
RNA and other sensitive materials are kept such as Primers and Ladders used in gel
electrophoresis.

Fig 17: Low temperature freezer

17. Water bath: Used to incubate samples in water at a constant temperature over a
period of time.
 Fig 18: water bath

3.1.2 Experience gained in molecular biology laboratory/ techniques used in molecular

biology lab.

1. Reagent preparation and uses

2. Sample collection and storage
3. DNA extraction/isolation
4. DNA quantification and qualitation using Nanodrop spectrophotometer
5. Amplification of DNA using polymerase chain reaction (PCR)
6. Gel electrophoresis of isolated DNA
 Reagent preparation and uses

 70% Ethanol
Calculation for preparation of 400ml of 70% ethanol from absolute (100%) ethanol.
Formular: C1V1=C2V2
C1=100%
V1=?
C2=70%
V2=400ml
V1 = C2V2÷C1 =70×400÷100
V1=280ml
H2O needed 400ml-280ml=120ml
Therefore, 280ml of absolute ethanol was added to 120ml of H2O to make 400ml of 70%
ethanol.

Procedure
 280ml of absolute ethanol was measured and poured into a clean measuring cylinder.
 Then, 120ml of H2O was added into the measuring cylinder containing the 280ml of
absolute ethanol.
 The solution was stirred to mix thoroughly and transferred into clean wash bottles.
 70% ethanol ready for use.

Uses of 70%ethanol
1. As effective disinfectant in the laboratory
2. Effective at killing bacteria and microbes.
3. For washing off residual salts from extracted DNA

 TBE (Tris Boric EDTA)

Preparation of 1×TBE for 2 litres
EDTA=0.74g (1litre) × 2=1.489(2litres)
Tris base=10.8g(1litre) ×2=21.6g(2litresl
Boric acid=5.5g(1litre) ×2=11.1g(2litres)

Procedure
1. The weighing balance was switched ON and set to zero
2. Clean crucible plate was placed inside and weighed 1.48g of EDTA, 21.6g of Tris base,
11.0g of Boric acid and were set aside.
3. A known volume of H2O was added into a beaker and stirred using magnetic stirrer.
 4. EDTA was added into the beaker and allowed to stir until dissolved.
Note:
Reason EDTA was added first is because it takes time to dissolve and it also act as a chelating
agent by binding to the salts added.
5. The pH of the solution was measured. (pH=8.0)
Note: add sodium hydroxide to increase pH or hydrochloric acid to reduce pH.
6. Tris base was added, followed by Boric acid and stirred till completely dissolved.

7. H2O was added to make up the required litre and stirred to obtain homogenized
mixture.
8. 1×TBE was transferred into clean bottles.
Note:

 Bottles were not filled to the brim.

 Bottles containing TBE were sterilized and allowed to cool before storing.
TBE ready for use.

Sample collection
Samples are collected from;

 Plant.
Note: Samples are best extracted from the actively dividing part which is the meristematic
region ie the young part of plant of interest.
 DNA extraction/isolation
This is the extraction or isolation of deoxy ribonucleic acid from sample of interest.
Method: CTAB Protocol (CetylTrimethyl Ammonium Bromide)
Steps involved: Lysis, precipitation, purification and elution.
Procedure for DNA extraction from sorghum seed

Lysis

 Sorghum seeds were collected from seed gene bank.

 Mortar and pestle were preheated at 65°c for 20 minutes.
 Lysis/extraction buffer was made ready for use by mixing 800Nl of beta
mercaptoethanol to 100ml of 10×ctab and placed in the water bath.
 0.3g of sample was weighed and crush in the mortar until it formed slurry.
 800Nl of extraction buffer was added to the finely crushed sample to form paste.
 The paste was poured into 1.5ml eppendorf tube and transferred into the water
bath for 20 minutes.
 The volume in the eppendorf tube was estimated and equal volume of SEVAGE
(chloroform and isoamyl alcohol in ratio 24:1) was added. Then the tubes were
shaked and opened consequently to allow gas escape.
 Tubes were transferred to orbital shaker for 1hr at 45rps (resolution per minute).
 Then spinned for 5 minutes at 10,000rpm using centrifuge.
 The gaseous supernatant was carefully pipetted out and dispensed into a new sterile
1.5 eppendorf tube.

Precipitation
The purpose of precipitating the DNA was to solidify it in a solution. Procedures include;
1. 500Nl of ice cold ethanol was added to the gaseous supernatant, rocked gently and
transferred into the freezer overnight.
Note: Leaving the sample overnight yielded more concentration of DNA but with lots of
impurities.
 Purification
The reason for this step was to wash any residual salt off from the extracted DNA. Procedure
includes;

 The sample kept in the freezer overnight was centrifuged for 5 minutes at 10,000rpm.
 Decanted.
 Equal volume of 70% ethanol was added and centrifuged for 2 minutes at 10,000rpm.
 Decanted.
 The step was repeated above.
 Decanted.
 Step was repeated again but for 5 minutes at 10,000rpm.
 Decanted.
 Tubes were air dried at room temperature until completely dry.

Elution
This step involved dissolving the dry DNA in sterile water or low TE buffer.

 Using a micropipette, 200Nl of nuclease free water (sterile water) was added into the
eppendorf tube containing the dry DNA sample.
 The dissolved sample was stored in – 40°c freezer for use in wide variety of downstream
analysis.

DNA quantification and qualitation using Nanodrop spectrophotometer

Nanodrop spectrophotometer is an equipment designed to quickly and easily measure nucleic
acid (DNA/RNA) concentration in sample of 1 microliter (1µL). Nanodrop spectrophotometer
operates on the Beer-Lambert principle which states that there is a linear relationship between
the concentration and absorbance of a solution which enables the concentration to be
calculated by measuring its absorbance.
Procedure used for quantifying DNA using Nanodrop Spectrophotometer

 The spectrophotometer and the connected PC was plugged to a power source.

 The pedestal was cleaned using sterile H20
 Nanodrop software was opened and nucleic acid application selected.
 Using a small volume calibrated micropipette, standardization/blank measurement was
performed by dispensing 1NL of sterile H20 (or low TE buffer, double distilled H20) unto
the lower optical pedestal. The lever arm was lowered and “Blank” selected in the
nucleic acid application.
 Once the blank measurement was complete, the pedestal was cleaned using paper
towel.
 1NL of DNA sample was dispensed unto the lower optical pedestal and the lever arm
was closed. Note; the sample only needed to bridge the gap between the optical surface
for measurement to have been made because the measurement was volume
independent.
 The sample identity was written on the space provided

 “Measure” was selected in the application software. The software automatically

calculated the DNA concentration in ng/µl by measuring the absorbance and purity
ratios at A260nm/A280nm.

Amplification of DNA using polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) is a technique used to make a segment of DNA into millions of
copy in-vitro. The technique was developed by Nobel Laureate Kary Millis in 1983

Applications of PCR
Some applications relying on the technology of PCR include the following:
1. DNA sequencing.
2. DNA fingerprinting.
3. Forensics.
4. Detection of bacteria or virus.
5. Diagnosis of hereditary disease.

Components of PCR
1. DNA template: This contains the region of the DNA fragment to be amplified (usually a
conserved gene or a gene of interest).
2. Primers (forward and reverse): Primers are short, single stranded DNA
(Oligonucleotides) sequences, which are complementary to the sequences at the end of
the target sequences to be amplified. They include the forward and reverse primer.
They are short, artificial DNA strands.
3. Nucleotides/dNTPs: These are deoxy nucleotide triphosphates required for extension of
the growing DNA strands
4. Taq polymerase: This is a type of thermostable DNA polymerase which can work at
higher temperature. Taq polymerase extends the primers and synthesizes copies of the
target DNA replication.
5. Co-factor (Mgcl2): Enhances the activity of Taq polymerase which in turn increase the
amplification rate of DNA.
6. Nuclease free H20 or double distilled H20: This is used to make up to the final volume of
the sample. Also used in other to dilute the concentration of the reagents to the proper
final concentration.

Procedure for preparation of reaction mixture

1. 25 eppendorf tubes were labeled and placed in an ice-rack.
2. Using a calibrated micropipette, 1NL of template, 1NL of forward primer, 1NL of reverse
primer, 10NL of mastermix and 8NL of Nuclease free H20 was dispensed into each
labeled 1.5 eppendorf tube.
3. Tubes were loaded into the thermal cycler/PCR machine.
4. After completion of 35 cycles, tubes were removed from the PCR machine and placed in
ice-rack.
5. Amplicons (amplified DNA) were stored in – 40°c freezer until ready for gel
electrophoresis.
Stages of PCR
1. Denaturation: Occurred at 94°c. Here, the reaction was strongly heated to
separate/denature the DNA strands. This provided single-stranded template for the next
stage.
2. Annealing: Occurred at 52°c. Here, the reaction was cooled so the primers could bind to
their complementary sequence on the single-stranded template DNA.
3. Extension/Elongation: Occurred at 72°c. Here, the reaction temperature increased so
Taq polymerase extended the primer, synthesizing new strands of DNA.

Gel electrophoresis of Amplicons (amplified DNA)

Gel electrophoresis is a technique that uses the principle of electrolysis to separate DNA
fragments based on their sizes and charges. An electric current is used to move molecules to be
separated through a gel.
Materials needed to run gel electrophoresis
Running buffer (1×TBE), 1.8% of agarose powder, gel stain (safe view stain), Amplicons
(amplified DNA), electrophoresis tank & current, DNA ladder etc.

Procedure for running gel electrophoresis

1. 2.7g of agarose power was weighed using sensitive weighing balance and poured into a
conical flask.
2. 150ml of 1×TBE was measured using measuring cylinder and poured into the conical
flask containing agarose powder.
3. The content was melted by heating in microwave.
4. And allowed to cool to 50°c. While cooling, apparatus was set up (gel casting tray with
comb).
5. 20NL of safe view stain was added.
6. Content was carefully shaked to mix thoroughly and poured immediately from one end
of the casting tray and allowed to solidify.
7. The gel combs were removed carefully
8. The casting tray containing the solidified gel was placed into electrophoresis tank
containing running buffer. The gel was positioned so that the chamber wells were
closest to the negative electrode of the chamber.
9. 5NL of Amplicon was loaded into the gel chamber well.
10. DNA ladder was added which served as reference.
11. Electrophoresis tank was connected to a power source and allowed to run at 101volt
and 90 ampere for 45 minutes.
After running the gel, the DNA fragments was viewed using UV transilluminator showing the
migration of DNA fragments from the negative charge electrode pole to the positive charge
electrode pole.
 Fig 19: Result of gel electrophoresis

The heaviest DNA strands was near the wells and the lightest was at the opposite end. The
DNA ladder served as a reference to measure the size of the DNA fragments.
3.2 Tissue culture laboratory
The Tissue Culture unit is a sub-unit of the Biotechnology Department in the National
Centre for Genetic Resources and Biotechnology. They carry out ex-situ conservation of plants
genetic resources.
What is Tissue Culture?
This is the in-vitro propagation of plant in a suitable media under aseptic, controllable and
artificial condition.
Principles of Plant Tissue Culture

 Totipotency
 Plasticity

Totipotency: The ability of a single plant cell to grow, divide, and differentiate into an entire
plant.
Plasticity: Is the abilities of plants to change their metabolism, growth and development in
order to best suit their environment.

Terms commonly used in plant tissue culture

Explant: A plant organ or piece of tissue used to initiate a culture.
Culture: Growing cells, tissues, plant organs, or whole plants in nutrient medium, under aseptic
conditions e.g. cell culture, embryo culture, shoot-tip culture, anther culture.
Contaminants: In tissue culture it refers to the micro-organisms (Bacteria, Fungi), which may
inhibit the growth of cells or tissues in culture.
Node: A region on the stem from which a leaf bearing an axillary bud arises.
Meristem: A localized group of actively dividing cells, from which permanent tissue system i.e.
root, shoot, leaf and flower are derived. Apical meristem is located at the apices of main and
lateral shoots.
Regeneration: In tissue culture, a morphogenetic response that results in the formation of new
organs, embryos or whole plants from cultured explants.
Dedifferentiation: The phenomenon of mature cells reverting to meristematic state to produce
callus is dedifferentiation. Dedifferentiation is possible because the non-dividing quiescent cells
of the explant, when grown in a suitable culture medium revert to meristematic state.
Redifferentiation: The ability of the callus cells to differentiate into a plant organ or a whole
plant is regarded as redifferentiation.
Types of tissue culture
1. Meristematic tissue culture
2. Node tissue culture
3. Embryonic tissue culture
4. Proplasm tissue culture

Applications of plant tissue culture

Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture.
Applications include:
1. The commercial production of plants used as potting, landscape, and florist subjects,
which uses meristem and shoot culture to produce large numbers of identical
individuals.
2. To conserve rare or endangered plant species.
3. A plant breeder may use tissue culture to screen cells rather than plants for
advantageous characters, e.g. herbicide resistance/tolerance.
4. To cross distantly related species by protoplast fusion and regeneration of the novel
hybrid.
5. To cross-pollinate distantly related species and then tissue culture the resulting embryo
which would otherwise normally die.
6. Certain techniques such as meristem tip culture can be used to produce clean plant
material from virused stock, such as sugarcane, potatoes and many species of soft fruit.
7. Production of identical sterile hybrid species can be obtained.
8. Large scale production of artificial seeds through somatic embryogenesis.

Advantages of plant tissue culture

1. For conservation of plant
2. Mass propagation of plants
3. Production of disease free plant
Disadvantages
1. Expensive technology
2. High skill personnel needed
3. Contamination problem
Table 1: Materials used in plant tissue culture
 OTHER WARES PLASTIC STAINLES GLASS
WARES S WARES WARES

Plasticine Hot plate Buckets Test tubes

PH metre Autoclave Tray Blade Measuring
holder cylinder
Oven Refrigerator Bowls Cannister Petridish
Foil Spatula Funnel Forcep Beaker
Pressure Water Plastic Conical
pot distiller measuring flask
cylinder
Dessicator Reagents Sterilizer Pipette
Surgical Masking Plastic Reagent
blade tape petridish bottles
Water Compound Media
deionizer microscope bottles
Laminar Magnetic Flat
flowhood stirrer bottom
flask
Waste bin Trolley Glass
stirrer
Hand Face mask
glove

3.2.1 Techniques used in plant tissue culture

1. Glassware preparation, sterilization and Purification of water.
 2. Media preparation.
3. Ex-plant collection and disinfection.
4. Transfer section (inoculation/culturing and sub culturing).
5. Growth room (growth of cultivated materials up to maturity).
6. Postflask management (hardening of plant/screening/quarantine rooms).

Glassware preparation
Glassware preparation was done by soaking glasswares in a big tank containing soduim
hypochrorite (Jik) and liquid soap for 2hrs before washing. After washing, was rinsed under
running water. Glasswares were subjected to double sterilization (wet and dry sterilization)
using the autoclave and hot air oven, after which they were stored in a sterile environment,
ready to be used.
Note; contaminated glasswares were soaked overnight after decontaminating them using
pressure pot before washing.

Media preparation
Procedure for preparation of yam regeneration media
1. Media preparatory room was disinfected using 70% ethanol.
2. Distilled water was made available and ions present in the water was removed by using
water deionizer.
3. A known volume of deionized water was added into a beaker and placed on magnetic
stirrer with the magnetic bar in the beaker.
4. The various media constituents (Macro and Micro nutrients, Iron, EDTA, Carbon source
etc) was weighed and poured into the beaker respectively.
5. After the constituents have been added, the pH electrode was dipped inside the media
to determine the pH of the media (5.7).
Note: If the pH was below 5.7-5.8, NaOH a base would have been added to increase the
PH. While, if the pH was above 5.7-5.8, HCL an acid would have been added to reduce
the PH.
6. Agar was added and melted using microwave.
7. Using a syringe, 50ml of the media was dispensed each into test tubes.
8. Media was sterilized using autoclave for 15 minutes before being stored in the growth
room till ready to be used.

What is media
It is a nutritional medium prepared in standard concentration to enhance the growth and
development of plants.
Types of media

 MS – Murashige and Skoog (1962)

 WPM – Woody Plant Medai
 VGA – Vitamins and Gibberllic Acid etc.
Basic constituent of media
 1. Macro nutrients (stock 1)
2. Micro nutrients (stock 2)
3. Iron and EDTA (stock 3)
4. Vitamins (stock 4)
5. Carbon source
6. Plant growth regulator (PGR)
7. Gelling agent/solidifier
8. Activated charcoal (optional).

Ex-plant collection and disinfection

Collection of vegetative part of plants was from field gene bank. Note; ex-plants are best
collected from the meristematic part of the plant ie the actively dividing part.
Disinfection procedure

 Ex-plant was washed thoroughly under running water using liquid soap to eliminate
surface dirt and thrice rinsed with distilled water after which it was soaked with 70%
ethanol for 5 minutes and thrice rinsed with distilled water
 After that, explant was soaked with sodium hypochlorite for 10 minutes and thrice
rinsed with distilled water.
 Ex-plant was finally placed on a sterile petri dish and transferred to the culture room for
inoculation.

Transfer section (inoculation/culturing and sub culturing)

Inoculation/culturing
This is the transfer of the ex-plant to a culture medium.
Procedure in culturing

 Laminar flow hood was switched ON and disinfected using 70% ethanol and swabbed
with cotton wool.
 Two petri dishes containing 70% ethanol was set.
 Culture vessel containing the already sterile prepared media was arrange neatly inside
the flow hood and sprayed with 70% ethanol.
 Hands were disinfected with 70% ethanol.
 Spirit lamp was lit, forceps flamed and allowed to cool.
 Explants were excised using sterilized petri-dish, forcep and blade suspended in a blade
holder.
  Explants were inoculated into the media and transferred to the growth room for
regeneration.
 Working area was cleaned and flow hood switched off.
Sub-culturing
This involved transferring the regenerated plantlet from its previous media into a new media
which could be proliferation media.
Types of Sub-culturing

 Nodal cutting for yam

 Pineapple stem subculture

Acclimatization/post flask management

Post Flask Management
Post flask Management is a technique which involves acclimatization, hardening or weaning of
tissue cultured plantlets after they are removed from culture container until finally established
on the field. This is an intermediate stage (transition) between the culture containers and the
field.
Post flask management technique can be described under five main stages:
1. Plantlets preparation: Plantlets to be acclimatized must have well defined shoots and
roots. It must be free from any forms of mechanical damage and contamination.
2. Safe handling of plantlets: Tissue culture plantlets are very tender, fragile and sensitive
to shock. Therefore optimal care is required in handling it, to prevent damage prior
transplanting.
3. Preparation of transplanting kits: The materials /medium used for transplanting must
be sterile to prevent contaminations, medium must be in the right proportions.
4. Transplanting: Plantlets are labeled with plastic tags prior to its removal from the
culture Vials. The plantlets removed are washed in a container of water in other to wash
off the media on the roots to discourage microbial growth. The roots are immersed
completely and covered with sterile soil medium then kept inside a white polythene
bag, watered, tied properly and then kept inside acclimatization chamber to avoid
wilting due to possible evapo-transpiration in the outside environment.
5. Releasing of humidity chamber: The TC plantlets are kept in the acclimatization
chamber for an average of one to two weeks before chamber is opened to release the
 humidity. The length of period at which plantlets are retained in the chamber depend
largely on the performance of plantlets.
6. Potting/ Field establishment: When plantlets has fully acclimatized, they then undergo
a stage of weaning called potting” whereby soil compositions are prepared and potted
in black polythene bags prior potting of plantlets. And when the plantlet has fully
developed to seedlings it’s then transferred to the field for establishment.
Importance/Advantages of Post flask Management to Tissue culture Plantlets.

 To create high level of humidity for the survival of in vitro plantlets.

 It’s a transition stage that enhances gradual adaptation of plantlets from in vitro to ex
vitro condition.
 It’s prevents plantlets from abiotic stress (low humidity, direct sunlight, wind, heavy rain
drops).
 To prevent dehydration of plantlets in an in vitro condition.
 To provide an absolute establishment of 100% survival of the plantlets for field
establishment.
NOTE: Certain things are usually looked into during the process or stage of acclimatization. For
instance;
The soil used must have the following components;

 Top soil.
 Stone dust or river sand.
 Coconut fibre (grinded).
TOP SOIL
It provides the plantlets rigidity or firmness when in the acclimatization medium.
STONE DUST/RIVER SAND
It serves as a permeable membrane for the medium. This is because stone dust or river sands
are porous in nature.
COCONUT FIBRE/HUSK
It softens the rigidity of the top soil since the plantlet’s roots are pseudo.
NOTE THAT:
The component of these acclimatization medium was added in several ratio of either 3:2:5 or
sometimes 3:5:5 respectively. Only fully established in vitro plantlets are weaned out for
acclimatization.
Acclimatization is carried on an already established cultured plantlet with the following
stipulations;
PROCEDURE

 The already established plantlets were collected from the growth room.
 The plantlet’s were removed from media vessel and rinsed carefully in water.
 Some polythene plant bags were filled up with the specially prepared sterile soil that has
been oven dried for about an hour at 250°C.
 Plant let’s were inoculated into the sterile soil filled plant bags before placing them in an
acclimatization tray.
 The plantlets were sprayed with distilled water before been placed in transparent
polythene and hanged in the acclimatization chamber.
 After 7 days, the polythene bags were punctured weekly for easy air passage, watering
and acclimatization.
The plantlets stayed for twenty one days in the chamber before finally being transported to the
screen house.
AIM OF ACCLIMATIZATION.

 Acclimatization is aimed at weaning about 100% of the fully established plantlets from
the in vitro to the ex vitro.
 To mass produce plants that is disease free.
 Acclimatization is an intermediary stage between the in vitro and the ex vitro nurturing
of plants. It basically serves as an adaptation system or ground for fully established
weaned in vitro plantlets.

USES OF ACCLIMATIZATION

 It helps in providing 100% survival rate in plantlets.

 It aids in attaining high relative humidity condition in ex vitro condition.
 It helps in hardening of the plant tissues (xylem and phloem)- it helps in re-establishing
the plantlets with rigid and well established tissues.
 It helps produce true-to-type plantlets (this is achieved by totipotency, plasticity and
Darwin’s theory of Common Descent-“like progenitor (parent) like progeny (offspring)”.
FACTORS AFFECTING ACCLIMATIZATION

 Extreme weather condition.

 High degree of contamination from the in vitro culturing.
 Fig 20: Acclimatization chamber.

SCREENING (SCREEN HOUSE)

This is where the plantlets were next transported to for continuous growth and adaptation to
the external environment. In the screen house, the plantlets were placed in a more comfortable
soil filled, durable and water containing polythene bags. The plantlets were watered daily, until
they fully elongated and well established for transplanting to the field finally. The screen house
is popular known as the green house or the nursery- plantlets are dropped in several pots for
establishment.

Fig 21: Screen house.

FIELD
This was the final stage in tissue culture processes because the plantlets were planted in the
field after screening has been carried out. Plants are them cultivated after all these several
 stages have been administered from the laboratory to the field where the plants are finally
catered and cared for.

CHAPTER FIVE

4.0 GLOSSARY OF WORDS AND INDUSTRIAL EXPERIENCES

LEARNT

GLOSSARY OF WORDS

Accession Number: This is the specific identification number of each

germplasm (seed) in the seed gene-bank.

Genebank: It is a facility or place that collects, catalogues and stores

genetic resources.

Germplasm: They are genetic resources such as seeds, tissues etc. that
can serve for the purpose of plant breeding.

Characterization: It is the determination or grouping of certain

morphological variables or expression of plant such as plant height, plant
colour, root length, seed size etc.

Orthodox Seeds: They are seeds whose moisture content can be reduced
to an appreciable level without losing their seed viability.

Recalcitrant Seeds: These are seeds whose moisture content cannot be

reduced to an appreciable level without losing their seed viability.
Mulching: Mulching is an horticultural practice that involves spreading the
a layer of material on the ground around plants to protect their roots against
heat, cold and loss of water.

Seed Treatment: It is a biological, physical or chemical agent applied to

seeds to help protect and improve the establishment of healthy crop.

Amplicon: It is the product of chromosomal DNA that has been amplified

or replicated using a thermocycler.

Ethylenediamine Tetraacetic Acid (EDTA): It is a reagent used in DNA

extraction that serves as a chelating agent.

Sevage: It is a reagent used in DNA extraction that contains the

combination of Chloroform and Isoamyl alcohol.

Beta-mercaptoethanol: It is a strong reducing agent that helps to clean

tannins and other polyphenols present in the crude plant extract.

Tris-EDTA Buffer: It is a reagent used in DNA extraction that functions by

solubilizing DNA, while protecting it from degradation.

Acclimatization: This is the process of taking In-vitro plants to a new

environment, in order to adapt the plant plant to an external environment.

Explant: Explant is a cell, organ or piece of tissue which has been

transferred from a plant to a nutrient medium.

Nursery: A nursery is a portion of agriculture where plants are propagated,

nurtured, grown, and sold out to the home garden or commercial purpose.
In-situ Conservation: This is the conservation of a plant species in its
natural habitat and the maintenance and recovery of viable population of
plant species in their original place.

Ex-situ Conservation: This is the conservation of a plant species outside

its natural habitat and the maintenance and recovery of viable population
outside their original place.

INDUSTRIAL EXPERIENCES LEARNT

During my 24 weeks of Students Industrial Work Experience Scheme

(SIWES), I acquired the following experiences:

I gained experience in handling and operating various equipment such as

Moisture meter, Micropipette, Centrifuge, Thermocycler, Autoclave and
Nanodropspectrophotometer.

I was able to carry out viability test on Orthodox seeds with little or no
supervision.

I was able to extract the DNA of maize leaf (Zea mays) and guinea corn
seed (Sorghum bicolor) with little supervision.

I was able to check for the DNA quantification of extracted DNA using
Nanodropspectrophotometer with no supervision.

I learnt the steps involved in gel electrophoresis.

I learnt the preparation of Yam Proliferation Media (YPM) and Yam

Regeneration Media (YRM).

I was able to collect data on occurrence or incidence of Fall Armyworm

(Spodoptera frugiperda).
3.3 SEED GENE BANK LABORATORY
This Unit has a lot of responsibilities of acquisition, managing, conservation, and utilization of
orthodox seeds. Orthodox seeds are seeds whose moisture content can be reduced to an
appreciable level without destroying the seed (embryo), and such seed can be stored for a long
period of time without losing its viability during ex-situ conservation.
3.3.1 Activities carried out at seed gene bank unit

 Exploration and collection.

 Registration of germplasm.
 Cleaning , drying, threshing,
 Seed viability test, moisture content determination.
 Seed conservation.
 Regeneration and multiplication of germplasm.
 Documentation of germplasm.
 Seed distribution to researchers.
  Varietal release. Etc.
Fig 22: Germplasm flowchart

Source of the Gene Bank Germplasm:

 From research institutes: Other research institutes bring germplasm to the gene-bank
for proper conservation.
 Exploration and collection: The centre researchers do go on genetic exploration to

search for new genetic resources or endangered resources (germplasm) and conserve
them for sustainable utilization.
 Donation: The centre gathers resources via donation. Scientist both in and out of the
country do donate germplasm to the centre to conserve them appropriately. After
collecting germplasm the next activity in the gene bank is registration.
Germplasm Registration: These include the passport data and the accession number.
Whenever the centre scientific officers go on exploration and collection they go with a booklet
called the passport data which contains some vital information such as collection location,
collector name, date of collection, vernacular cultivar, village, state, precise location, collection
number, latitude, longitude, description notes, material, and frequency.
Accession number: This is the identification number of each germplasm in the seed gene-bank.
It’s the mines of identification of the germplasm in the gene-bank. Each germplasm have a
specific identification number (accession number).
Type of Germplasm
Orthodox germplasm, recalcitrant germplasm and the Intermediate germplasm.
Orthodox germplasm. These are seeds which moisture content can be reduced to an
appreciable level without destroying or damaging it, and the orthodox germplasm can be
stored for a long period of time without losing the seed viability and vigour, and this is the type
of germplasm that we conserve in NACGRAB gene-bank i.e. they can withstand both freezing
and drying, examples include sorghum (Sorghum bicolum), rice (oriza sativum), cowpea (Vigna
panicunata).
Recalcitrant germplasm. This is the exact opposite of orthodox germplasm, cause they cannot
be stored for a long period of time under any temperature without losing their seed viability
and vigour, and their moisture content can’t be reduced to any level without destroying them
i.e. they can neither withstand freezing nor drying, example include mango (Mangifera indica),
pawpaw (Carica papaya), Guava (psidiun guajava).
Intermediate germplasm. They are neither orthodox nor recalcitrant it’s an intermediary
between the orthodox and recalcitrant, It’s said to be the balancing point between the two
cause sometimes it tends to behave like orthodox and sometime it’s recalcitrant e.g. coffee.
Conservation methods: long term gene-bank for base collection (-20˚C / 10% R.H), short term
gene-bank for active collection (18˚C / 40% R.H), botanical garden, cryopreservation bank, DNA
bank.
After identifying the type of germplasm collected the next activities is: cleaning, drying,
threshing and separation.
Cleaning: This is the process or act of removing dirt on the collected germplasm or the
purification of germplasm.
Drying: This is the process of making germplasm free from liquid or moisture, it’s also the
process of reducing the seed moisture content to an appreciable level without destroying the
seed / germplasm. Drying methods: sun drying, using of seed dryer, desiccator (a closed glass
vessel containing a desiccant (such as silica gel or carbon oxide) used for keeping germplasm
dry. Moisture percentage of the stored seed is usually 11%.
Threshing and separation: This is the separation of grain from the straw or husks by mechanical
beating, with a flail or machinery (threshing machine).
After seed purification and drying next thing is to test on viability, dormancy, and quality.
Viability test: Viability is the process of ascertaining wither a seed is alive or not, wither seed is
viable(able to live and develop). This test is usually done before keeping or storing germplasm
in the various storage environments (the gene-banks, and refrigerator).
Method followed: Filter papers were placed neatly in petri-dishes, then soaked with 20 ml of
distilled water, and the seeds were carefully placed on the soaked filter paper, before being
placed in an incubator at 20 ± 1˚Cfor 8-20days. Normal seedlings were counted according to
international rules of ISTA (1993). Germination percentage was calculated using the following
formula outlined by Krishnasamy and Seshu (1990)
: Germination (%) = Number of seed germinated × 100%
Number of tested seed
Dormancy in seeds
Dormancy: This is the inability of a seed to germinate or grow under a favorable condition e.g.
okra seed, rice i.e. if a seed is exposed to all necessary or favorable condition for its germination
and it refuses to grow that doesn’t mean the seed is not viable but dormant (in its resting or
inactive state) and there is need to break dormancy for such seed. There are several methods
for breaking seed dormancy such as; Scarification method, hot water treatment, Acid treatment
Scarification method: This is the act of scarifying or the removal of the seed coat which
constitute to the seed dormancy by preventing water from entering the seed. Scarification was
done with the use of a sharp object so as to make water passage into the seed easy. Instrument
used include; knife, blade, scissors, sandpaper.
Hot water treatment: This was carefully done by immersing the seed into hot water once and
air dried afterwards.
Acid treatment: Concentrated acid like hydrochloric acid (HCL) was used to break seed
dormancy, by immersing the seed in the acid for some minutes.
Characterization and evaluation: This involved the process of characterization and the
determination of certain variable or expression such as plant height, plant colour, root length,
shoot length, seed size, leaf chlorophyll test, leaf size etc.
Documentation: This involved keeping useful information (written amount of an idea) on
processed germplasm before being stored in the various storage environments.
 Chapter 4:
Conclusion

This report contains detailed knowledge/experience I acquired during my six months industrial
training in molecular biology laboratory, tissue culture and seed gene bank of NACGRAB. I was
exposed and equipped with practical learning of the theories being taught in school. Through
the SIWES internship, I have been able to acquire knowledge and more comprehensive
understanding about the working conditions in different departments in line with my course of
study. I have been able to gain all these experience and acquired relevant knowledge, not only
through direct involvement in task delegated to me, but through other aspects of training such
as interaction with colleagues and asking questions from my superiors.
4.1 Skills developed/experience gained
The industrial training gave me a lot of experience by exposing me to the practical aspect of the
theories I have been taught in school. I was able to extract genomic DNA from sorghum seed,
yam leaf etc and I was able to micropropagate different varieties of yam in-vitro under aseptic
conditions for regeneration. I also learnt how to culture, subculture and acclimatize yam
plantlets. And also carry out viability test on germplasm.
4.2 Challenges encountered during industrial training

 Difficulty in operating most of the equipments in the lab during first week of work.
 Unstable power supply which lead to delay in work.
 Had difficulty in performing excision on tissue for the first time.
 Contamination of cultured explants.
 Intolerance to Phostosin even with the aid of nose mask while fumigating germplasm
(seeds).

4.3 RECOMMENDATION
1. ITF should make monthly allowances available for the students in order to minimize
financial difficulties that may arise as a result of transportation.
2. The institution and ITF should provide places for industrial attachment for students.
3. Company of attachment should provide more safety equipments to prevent further
environmental and health hazards.

TECHNICAL REPORT
 ON

STUDENTS INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

UNDERTAKEN AT

NATIONAL CENTRE FOR GENETIC RESOURCES AND

BIOTECHNOLOGY

( NACGRAB )

P.M.B. 5382 MOOR PLANTATION, IBADAN, NIGERIA.

BY

ADALUMO AANUOLUWAPO OLAWALE

190410006

SUBMITTED TO

THE DEPARTMENT OF PLANT SCIENCE AND BIOTECHNOLOGY,

ADEKUNLE AJASIN UNIVERSITY, AKUNGBA-AKOKO, ONDO STATE

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD

OF BACHELOR OF SCIENCE (B.Sc) IN PLANT SCIENCE AND
BIOTECHNOLOGY.
August, 2022
 CERTIFICATION

This is to certify that the SIWES internship and this report was carried out
and written by ADALUMO AANUOLUWAPO OLAWALEof the Department
of Plant Science and Biotechnology with matriculation number
190410006 as meeting part of the requirement for the award of Bachelor of
Science B.sc (Hons) Degree in Plant Science and Biotechnology.

_________________________ ______________________

STUDENT SIGNATURE/ DATE

_________________________ ______________________

SIWES CORDINATOR SIGNATURE/ DATE

_____________________ _________________

HOD SIGNATURE/ DATE

 DEDICATION

This technical report is dedicated to Almighty God for His mercy, favour and
protection throughout my SIWES internship.
 ACKNOWLEDGEMENT

My deepest gratitude goes to God for life given, preserved and sustained.

I acknowledge with unreserved gratitude, my HOD in person of Dr. Osekita,

my SIWES Coordinator, Dr. Akanji, my SIWES Supervisor, Dr. Ajayi and all
lecturers of the prestigious Plant Science and Biotechnology department for
their contributions to my academic pursuit.
I sincerely appreciate the Director of National Centre for Genetic
Resources and Biotechnology in person of Dr. S.E. Aladele, my industry
based Supervisor, Mrs. Jamaleddine, all staff of National Centre for Genetic
Resources and Biotechnology and my fellow SIWES students for making
my SIWES a successful one.

This acknowledgement will not be complete without appreciating my family

for their tireless support both spiritually, morally and financially throughout
my SIWES internship.

TABLE OF CONTENT

TITLE PAGE i

CERTIFICATION ii
DEDICATION III
ACKNOWLEDGEMENT iv

TABLE OF CONTENT v

EXECUTIVE SUMMARY vi
CHAPTER 1: INTRODUCTION AND OBJECTIVE OF REPORT 1

1.1 Introduction 1

1.2 Objective of Report 1

CHAPTER 2: NATURE OF BUSINESS/LITERATURE REVIEW OF INDUSTRY 5

2.1 History of Company 5

2.2 Company's Mission and Vision statement 6

2.3 Management Structure of the Company 6

2.4 Brief write-up on the Industry in Nigeria/Market Service Situation 7

2.5 Brief Discussion on the Economic Environment in which the Company Operate 9

CHAPTER 3: DUTIES/ WHAT I LEARNT (MY EXPERIENCE) 10

3.1 Brief Description of Work done 10

3.2 Skills Developed and Techniques Learnt 63

3.3 Practical Challenges Faced at Work 64

3.4 Theoretical Principles Learnt at Work 64

3.5 Specific Contribution Made 65

3.6 The Future of the Company in the Nigeria Economy 66

CHAPTER 4: RELEVANCE OF INDUSTRIAL EXPERIENCE GAINED TO MY COURSE

OF STUDY 67

4.2 Relevance of Industrial Experience Gained to my Course of Study 67

CHAPTER 5: GLOSSARY OF WORDS AND INDUSTRIAL EXPERIENCE LEARNT 68

CHAPTER 6: RECOMMENDATION 72

CHAPTER 7: CONCLUSION 73

EXECUTIVE SUMMARY

The Students Industrial Work Experience Scheme (SIWES) is a

training designed and put forward to expose and prepare students for what
they are to encounter after graduation. The main goal of this scheme is to
expose the students to different methods and experience needed for them
to carry out jobs relating to their field of study effectively and efficiently.

The training is also designed to expose students to the practical

aspect of their course of study, what they could not be taught in school as
well as the needed experience in handling equipment, machinery and skills
that are provided and those that cannot be provided by their educational
institutions but are available in the organization in which they were
attached.

This report has been prepared and divided into seven chapters for
the sole purpose of making it clear to the readers.

Chapter one of this report shows the introduction and objective of the
report, nature of industry, history of the organization in which the SIWES
has been carried out. Chapter two comprises of the nature of the business
and literature review. Chapter three explains the duties, as well as what I
learnt, my challenges, theoretical principles learnt and the contribution I
made to the organization. Chapter four shows the relevance of the training
to my course of study, while chapter five talks about the glossary of words
and industrial experience learnt in the course of the internship. Chapter six
and seven of this report shows my recommendation to the organization and
a conclusion that gives the summary of my experience at National Center
for Genetic Resources and Biotechnology respectively.
 CHAPTER ONE
1.0 INTRODUCTION AND OBJECTIVE OF REPORT

1.1 INTRODUCTION

The students Industrial Work Experience Scheme (SIWES) is the

accepted skills training programme, which forms part of the approved
minimum academic standards in the various degree programmes for all
Nigerian Universities. It is an effort to bridge the gap existing between class
and field, theory and practice of engineering and technology, science,
agriculture and other professional educational programmes in the Nigerian
tertiary institutions. It is aimed at exposing students to machine and
equipment, professional work methods and ways of safe-guarding the work
areas and the workers in industries and other organizations.

The minimum duration for the SIWES is 24 weeks (6months). The

scheme is a tripartite programme, involving the students, the Universities
and the Industries (employers of labour). This scheme is funded by the
Federal Government of Nigeria and jointly coordinated by the Industrial
Training Fund (ITF) and the National Universities Commission (NUC).

It is on this note that I, ATIMOKHALE DANIEL, participated in the

programme which commenced March 2022 to August 2022, at the National
Centre for Genetic Resources and Biotechnology, P.M.B. 5382 Moor
Plantation, Ibadan, Nigeria.

1.2 OBJECTIVE OF REPORT

The major aim and objective of the Students Industrial Work Experience
Scheme (SIWES) Technical Report is to give a brief description of work
experience(s) gained. This report also covers the major aims and
objectives of the Students Industrial Work Experience Scheme (SIWES)
which are:

To expose students to industrial environments and enable them develop

occupational competence for them to contribute their quota to the national
economy and scientific development after graduation.

It affords students of tertiary institutions the opportunity of being

familiarized and exposed to the needed experience in handling machinery
and equipment which are usually not available in the educational
institutions.

To allow students gain the technical experience on how to behave and

relate with people in working environment.

The scheme exposes students to industry based skills necessary for a

smooth transition from the class room to the world of work.

BODIES INVOLVED IN THE MANAGEMENT OF SIWES

There are five bodies that are involved in the operation of SIWES in
Nigeria. They include the following:

The Federal Government

The Industrial Training Fund (ITF)

The Collaborating Agencies (NUC, NBTE and NCEE)

 The Institution

The Employer

THE FEDERAL GOVERNMENT

The Federal government is charged with the following responsibilities;

Make fund available to the Federal Ministries of Industries to fund

the scheme.

Make it mandatory for all establishments to offer places to the

students for attachment.

THE INDUSTRIAL TRAINING UNIT (ITU)

To organize workshops, seminars and conferences on SIWES.

To compile the list of employers and available places for industrial

experience and forward such lists to the coordinating agencies.

To provide the logistic materials needed for the administration of the

scheme.

THE NIGERIAN UNIVERSITIES COMMISSION (NUC)

To establish SIWES coordinating agencies.

To appoint full time industrial coordinators to operate the scheme at

the agency level.

To approve SIWES master and placement lists and forward such

lists to ITF.
 To involve the minimum National aid program for supervised
Industrial activities for approved SIWES courses.

THE UNIVERSITY

To establish SIWES coordinating units and appoint departmental

and faculty coordinators within the institution.

To help secure placement of students on attachment with employers.

To organize orientation programme for students to prepare them for

their industrial training.

To assess the students’ performance and award grade accordingly.

To help in submitting the ITF forms to the training fund at the end of
the programme.

THE EMPLOYER

To accept students for Industrial Attachment as stipulated in ITF

Decree No. 47 as amended (1990).

To participate fully in the assessment of students by completing the

necessary instruments e.g. ITF form 8, logbook etc.

To allow students have access to their facilities.

To appoint an Industry-based Supervisor for students on attachment

 CHAPTER TWO
2.0 NATURE OF BUSINESS/LITERATURE REVIEW OF INDUSTRY

2.1HISTORY OF COMPANY

National Centre for Genetic Resources and Biotechnology

(NACGRAB) was established in 1987 by the Federal Ministry of Science
and Technology (FMS&T) in other to conduct research, gather data and
disseminate technological information on matters relating to genetic
resources conservation, utilization and biotechnology applications. The
Centre, backed by Decree 33 of 1987 regulates the seed, livestock and
fisheries industries through its Varietal Release Committees.

In January, 2004 the Centre was up-graded to the status of a

parastatal. However, during the reorganization of parastatal and agencies
under the Federal Ministry of Science and Technology in 2007, NACGRAB
was merged with National Biotechnology Development Agency (NABDA),
NACGRAB through its various programmes, has continued to contribute
significantly to the conservation of the rich genetic resources of the nation.
Programmes of the Centre include:

Conservation, preservation and maintenance of valuable plants and

animal genetic resources, for immediate utilization and posterity;

Development of animal genetic resources (poultry, snailry, fisheries etc).

Application of tissue culture for plant conservation and overall agricultural

development;

Servicing of the activities of the National Committee on Naming,

Registration and Release of Crops Varieties, Livestock Breed and
Fisheries;

Networking and coordinating activities in the development of capacities

in plant and animal genetic resources.

2.2 COMPANY’S MISSION/VISION STATEMENT.

MISSION

Nigeria is endowed with diverse biological heritage, which needs to be

maintained for the purpose of utilization and for posterity. NACGRAB is to
ensure the conservation and sustainable use of these rich biodiversity
through Research and Development.

VISION

A centre that is committed to the conservation of the rich Genetic

Resources of the nation, with a view of enhancing agricultural, economic
and social development.
2.3 MANAGEMENT STRUCTURE OF NATIONAL CENTRE FOR
GENETIC RESOURCES AND BIOTECHNOLOGY.

NACGRAB is headed by a Director/Chief Executive Officer ( CEO ).

He is responsible for daily operations at the Centre. He also oversees the
activities of various units at the Centre.

The Centre has a total of seven ( 7 ) units namely: Plant Genetic

Resources ( PGR ), Animal Genetic Resources ( AGR ), Biotechnology,
Administration, Accounts, Maintenance and Information and
Documentation, each unit has a head who oversees/coordinates the
operations of the unit and in turn reports directly to the Director/CEO. The
Centre has a staff strength of over 500 people.

ORGANISATIONAL CHART OF NACGRAB

2.4 BRIEF WRITE UP ON THE INDUSTRY IN NIGERIA / MARKET
SERVICES SITUATION

National Center for Genetic Resources and Biotechnology, Moor

Plantation, Ibadan is grouped under Research Institutions for the
development of Science and technology with a view to foster Agricultural
sector and National Food Security through several measures of
conservation of valuable genetic resources for sustainable utilization.
NACGRAB is established to conduct research to develop facilities for the
identification of national research need and priorities and to regulate the
utilization of genetic resources, genetic engineering and biotechnology and
for the training of technical and sub-technical personnel for the service in
the company.

NACGRAB is a core research and educational institution and was

established in 1987 by the Federal Ministry of Science and Technology
(FMS&T) to conduct research, gather data and disseminate technological
information. Services rendered by NACGRAB are strictly for study,
conservation and improvement of practices.

THE RESEARCH MANDATE

National Center for Genetic Resources And Biotechnology in Nigeria

market renders the following services via its mandate;

Exploration, collection, identification, evaluation, characterization, storage

and conservation of rich stock of both animal and plant germplasm
material.

National coordination of genetic resources programme and its sustainable

utilization.

Fostering relationship with other national satellites genetic research centres

located in research institutes, universities and polytechnics as well as the
international organizations and centres of programmes concerning genetic
resources and biotechnology application.
Arrest rapid erosion and loss in the country’s crop and animal genetic
resources caused by cultivation, urbanization, rural development,
desertification, national catastrophes etc.

Documenting appropriately the germplasm stocks held by the centre,

research institutes and relevant organizations.

2.5 BRIEF DISCUSSION ON THE ECONOMIC ENVIRONMENT IN

WHICH NATIONAL CENTRE FOR GENETIC RESOURCES AND
BIOTECHNOLOGY OPERATES.

NACGRAB'S programmes cut across several sectors under Nigeria

economic environment ranging from Science and Technology Development
down to Agricultural production and Food Security. To execute the mandate
effectively and ensure proper running of activities within the organization,
NACGRAB collaborate with indigenous and international research institute,
universities and biotechnological companies abroad [INGABA
Biotechnology]. Considering the location of NACGRAB, it is not the only
research in the area, other research institute that are few kilometers away
include: National Horticultural Research Institute [NIHORT], Institute of
Agricultural Research and Training [IAR & T] liaison with other relevant
international organization e.g. International Plant Genetic Research
Institute [IPGRI], Food and Agricultural Organization [FAO] and the
International Institute of Tropical Agriculture [IITA].

CHAPTER THREE
3.0 DUTIES / WHAT I LEARNT (MY EXPERIENCE)
3.1 BRIEF DESCRIPTION OF WORK DONE.

During the period of my SIWES internship at the National Centre for

Genetic Resources and Biotechnology, I was firstly posted to the Seed
Gene-bank unit, then to biotechnology department which comprises of
Molecular laboratory and Tissue Culture laboratory, and lastly Field Gene-
bank unit. In these four departments, I learnt and practically participated in
so many activities that are beneficial to my course of study.

SEED GENE BANK

Seed Gene-bank is one of the sections under Plant Genetic

Resources (PGR) in Nacgrab the other being Field Gene-bank. The two
gene-banks are of two different mandates in terms of seeds storage (Seed
gene-bank stores Orthodox seeds while Field gene-bank stores
Recalcitrant seeds) but with the same mandate based on the purpose of
conservation of genetic resources.

This Unit has a lot of responsibilities of acquisition, managing,

conservation, and utilization of Orthodox seeds. Orthodox seeds are seeds
whose moisture content can be reduced to an appreciable level without
destroying the seed (embryo), and such seeds can be stored for a long
period of time without losing its viability during ex-situ conservation.

ACTIVITIES CARRIED OUT AT SEED GENE BANK UNIT

Exploration and collection.

Registration of germplasm.

Cleaning, drying, threshing and separation.

Seed viability test.

Moisture content determination

Seed conservation.

Regeneration, characterization and multiplication of germplasm.

Documentation of germplasm.

GERMPLASM FLOW CHART

Source of t

Sources of Germplasm:

1. Research Institutes: Research Institutes such as Forestry Research

Institute of Nigeria (FRIN), International Institute of Tropical Agriculture
(IITA), Cocoa Research Institute of Nigeria (CRIN) etc bring germplasm to
the gene-bank for proper conservation.

2. Exploration and collection: The centre researchers go on genetic

exploration to search for new genetic resources or endangered resources
(germplasm) and conserve them for sustainable utilization.

3. Donation: The centre gathers genetic resources via donation. Scientists

both in and out of the country donate germplasm to the centre to conserve
them appropriately.

After collecting germplasm (seeds), the next activity is registration of such

germplasm

Germplasm Registration: This includes passport data and accession

number. Whenever the centre scientific officers go on exploration and
collection, they go with a booklet called the passport data which contains
some vital information such as collection location, collector name, date of
collection, vernacular cultivar, village, state, precise location, collection
number, latitude, longitude, description notes, material, and frequency.
Accession number is the identification number of each germplasm in the
seed gene-bank. It’s the mines of identification of the germplasm in the
gene-bank. Each germplasm have a specific identification number
(Accession number).

Note: Germplasm is any living part of a plant that can be used for plant
breeding. Examples include seed, root, leaf, stem etc.

Types of Germplasm (Seeds): There are three major types of germplasm,

namely; Orthodox Germplasm (seeds), Recalcitrant Germplasm (seeds)
and the Intermediate Germplasm(seeds). Orthodox Seeds moisture
content can be reduced to an appreciable level without destroying or
damaging it, and the orthodox germplasm can be stored for a long period of
time without losing it seed viability and vigour, i.e. they can withstand both
freezing and drying, examples include Pearl millet (Pennisetum glaucum),
Maize (Zea mays), Guinea corn (Sorghum bicolor) etc. Recalcitrant seeds
are the exact opposite of orthodox germplasm; they cannot be stored for a
long period of time in any temperature without losing their seed viability,
and their moisture content can’t be reduced to any level without destroying
them i.e. they can neither withstand freezing nor drying, example include
mango (Mangifera indica), pawpaw (Carica papaya), Sausage tree (Kigelia
africana) etc. Intermediate seeds are neither orthodox nor recalcitrant it’s
an intermediary between the orthodox and recalcitrant, It’s said to be the
balancing point between the two because sometimes it tends to behave like
orthodox and sometimes like recalcitrant e.g. coffee.
After identifying the type of germplasm collected the next activities are:
cleaning, drying, threshing and separation.

Cleaning: This is the process or act of removing dirt on the collected

germplasm. It is also the purification of germplasm.

Drying: This is the removal of liquid or moisture from germplasm. It is also

the process of reducing the seed moisture content to an appreciable level
without destroying the seed / germplasm. Drying methods include sun
drying, use of seed dryer, desiccator (a closed glass vessel containing a
desiccant such as silica gel) used for keeping germplasm dry. Moisture
percentage of the stored seed is usually 11%.

Threshing and separation: This is the separation of grain from the straw
or husks by mechanical beating, with a flail or machinery (threshing
machine).

After seed purification and drying, next is to test on viability of the seeds

Viability Test: Viability test is the process of ascertaining whether a seed is

alive or not, whether the seed is viable(able to live and develop). This test
is usually done before keeping or storing germplasm in the various storage
environments (the gene-banks).

Materials for conducting seed viability test include: petri-dishes or

plastic container, seed master for counting, biochemical-incubator, tissue
paper, syringe and distilled water.

Method: The tissue papers (media) were placed neatly in the plastic
containers, and soaked with 8- 10 ml of distilled water depending on the
hardness of the tissue paper (medium), and the seeds were carefully
 placed on the soaked tissue paper, and
placed in an incubator at 20 ± 1˚Cfor 8days.
Normal seedlings were counted according
to international rules of ISTA (1993).
Germination percentage was calculated
using the following formula outlined by
Krishnasamy and Seshu (1990)

Germination (%)/ % Viability =Number of seed germinated × 100%

Number of tested seed

 Viability test on Sorghum seeds (Sorghum bicolor)

After checking the germination percentage or viability percentage of

the seeds, seeds that are not viable are discarded, for dormant seeds, the
dormancy is broken, seeds with low viability are taken to the field for
regeneration, while seeds with high viability are stored in the gene-bank
(Long-term and Short-term Genebank).

Dormancy: Dormancy is the inability of a seed to germinate or grow under

a favorable condition i.e. if a seed is exposed to all necessary or favorable
condition for its germination and it refused to grow, that doesn’t mean that
the seed is not viable, the seed is just dormant (in its resting or inactive
state) but there is need to break dormancy for such seed. There are
several methods for breaking seed dormancy such as: scarification
method, hot water treatment method, Acid treatment method etc.

Scarification method: This is the removal of the seed coat that do constitute
to the seed dormancy by preventing water from entering the seed.
Scarification is done with the use of a sharp object so as to make water
passage into the seed easy. Instruments are, knife, blade, scissors,
sandpaper.

Hot water treatment: This is carefully done by immersing the seed into hot
water just once and air-dry the seed.

Acid treatment: Concentrated acids such as hydrochloric acid (HCl),

TetraoxosulphateVI acid can be used to break seed dormancy, by
immersing such seed in the acid for some minutes.
Regeneration: Regeneration of seeds is carried out on seeds with low
viability. The seeds are taken to the field and then planted to get seedlings
with higher or better viability.

Multiplication: Multiplication of seeds is done on seeds with low quantity.

The seeds are either taken to the screen house or field and then planted to
get multiple seeds.

Characterization and Evaluation: During regeneration and multiplication

of seeds, characterization and evaluation is carried out. Characterization is
the determination of certain variables or expressions both qualitative and
quantitative expression of plant such as plant height, seed size, plant
colour, root length etc.

After taking characterization of the plant, freshly harvested seeds are

taken to the laboratory and then the moisture content of the seeds are
determined and the seeds are stored in the genebank.

Moisture Content Determination: According to Harrington's Thumb

Rule on Seed Storage which states that: For every 1% increase in
moisture content or a 5°C increase in storage temperature, the life of the
seed is reduced by half. Before storing seeds, it is mandatory the moisture
content of such seeds is checked because the moisture content of seeds
determines the shelf life of the seeds. This is done using instruments such
as Moisture meter, Moisture analyzer etc.

Seed Treatment: Seed Treatment is the biological, physical and chemical

agents applied to seeds in order to provide protection and improve the
establishment of healthy crops e.g Cowpea seeds (Vigna uniguiculata) are
 treated with fustocine to prevent weevil. Seeds
must be treated before storing them in the
genebank.

Packaging and Labelling: Packaging of seeds involves packing seeds

using packing materials such as envelope, aluminium foil, plastic etc while
labelling involves attaching certain information such as name of the seed
and accession number given to the seed.

Documentation: This involves keeping useful information about the seeds

before storing them in the genebank.

Conservation methods: After packaging, labeling and documenting the

germplasm(seeds), it is necessary the seeds are stored in a good
environment. Methods of conserving Orthodox Germplasm (Seeds)
include; long term gene-bank for base collection (-20˚C / 10% R.H), short
term gene-bank for active collection (18˚C / 40% R.H), botanical garden,
cryopreservation bank, DNA bank.

Recording the temperature and relative humidity of Long-term genebank

MOLECULAR LABORATORY
 The molecular laboratory is one of the sections under biotechnology
department in Nacgrab. During my third month as an intern, I was posted to
molecular laboratory which I spent one month and few days in the unit.

Molecular Biology: This is a branch of biology that deals with the study of
the structure and function of macro molecules (Nucleic acids and Proteins).
Molecular biology can also be said to the study of the genetic information of
plants, animals and microbes.

Advantages of Molecular Biology Technique

It is not influenced by the environment i.e. it is under controlled

environment.

It is not affected by the stages of development.

Traits observed are unlimited using various molecular markers.

Applications of Molecular Biology Technique

It is applicable in DNA fingerprinting.

It is used in forensic science.

It is also applicable in determining pathogens.

It is used in genetic diversity study.

Biosafety precautions in Molecular Laboratory

Put on you lab coat to avoid spillage.

Use hand gloves to protect yourself from contaminants.

Do not eat, drink or smoke in the laboratory.

Always wash your hands after you have engaged in practical work in the
laboratory.

Switch off your handset to avoid distraction.

Do not mouth pipette anything in the laboratory.

Report any accident to the lab scientist.

EQUIPMENT USED IN MOLECULAR LABORATORY

Micropipette: It is used to accurately and precisely measure small

volumes of liquid. It has various sizes with corresponding colours. The
yellow colour ranges from 20µl to 200µl with one decimal point, the grey
colour ranges from 0.5µl to 10µl with two decimal points and the blue
colour is just 1000µl without decimal point.

Set of Micropipette

Centrifuge: It is an equipment used to separate liquids or mixtures based

on their densities. In DNA extraction, centrifuge separates mixtures into
three layers namely; the Supernatant (dense), denatured protein(more
dense) and the organic components (most dense).
 Centrifuge

Thermocycler (Polymerase chain reaction machine): This is a machine

used for amplification or replication of a target region of DNA.

Thermocycler

Nanodrop spectrophotometer: It is an equipment used to check for the

quality (purity) and quantity (concentration) of isolated DNA and RNA.
 Nanodrop spectrophotometer

Vortex mixer: It is used to homogenize mixtures during DNA extraction.

Vortex mixer

Water bath: It is an equipment used to incubate samples in water at

constant temperature over a period of time.
 Water bath

Low/Ultra temperature Freezer: It serves as a genebank in storing

extracted DNA and reagents such as DNA ladder, master mix, TE/TBE
buffer etc. The freezer is as low as -40ºC hence it came isolated DNA for
some years without the DNA degrading.

Ultra temperature Freezer

Electrophoresis tank and power pack: It consists of gel tank, tray, gel
comb and electrophoresis power pack. They are used for agarose gel
electrophoresis in order to determine fragments of DNA sizes.
 A gel tank connected to power pack

Ultraviolet documentation: This is a machine used to deplay the result of

gel electrophoresis i.e it visualizes fragments of DNA. It has an ultraviolet
transilluminator, dark room to shield the external light source, which protect
the user from ultraviolet light exposure, and also a CCD camera for
capturing.

UV Documentation

ANALYSES IN MOLECULAR LABORATORY

The following are the various work done in molecular laboratory in
Nacgrab:

Reagents preparation

Sample collection

DNA extraction/ Isolation

DNA quantification

Polymerase Chain Reaction(PCR)

Agarose gel electrophoresis

REAGENTS PREPARATION

In extracting, quantifying and amplifying DNA, reagents are required.

One of the core reagents needed in the above analyses is Tris
ethylenediamine tetra acetic acid Buffer(TE Buffer) or Tris borate
ethylenediamine tetra acetic acid Buffer(TBE Buffer).

Preparation of 10× TE Buffer

Components required: 2%CTAB, 1%PVP, 100mM of Tris HCl, 1.4M of

NaCl, 20mM of EDTA and 1litre(1000ml) of distilled water

CTAB(Cethyltrimethylammoniumbromide)- 2%

2/100×1000=20g

PVP(Polyvinylpyrrolidone)- 1%
1/100×1000=10g

Tris Hydrochloric acid- 100mM

100×1/1000= 0.1M

Mole= mass/molar mass

Molar mass of Tris HCl= 157.6g/mol

Mass= mole×molar mass

Mass= 0.1×157.6= 15.76g

NaCl- 1.4M

Mass= mole× molar mass

Molar mass of NaCl= 58.6g/mol

Mass= 1.4×58.6= 82.04g

EDTA- 20mM

20×1/1000= 0.02M

Mass= mole× molar mass

Molar mass of EDTA= 372. 24g/mol

Mass= 0.02×372.24= 7.44g

Procedure:

Dissolve all components (20g of CTAB, 10g of PVP, 15.76g of Tris HCl,
82.04g of NaCl and 7.44g of EDTA) in 1litre(1000ml) of distilled water and
stir vigorously, then store in -40ºC freezer.
 SAMPLE COLLECTION

Sample collection is the gathering of any living part of the plant,

animal or microbe for DNA extraction and other Downstream analyses. The
parts of the plant collected as sample include; Seed, leaf, stem, root etc.

Steps in sample collection of plant

1. Identify the part of a plant you want to work with.

2. Target the apical or youngest part of the plant for collection. Reasons for
this are;

The youngest part of the plant contains actively dividing cells i.e. the
meristematic cells.

The apical or youngest part of the plant posseses less unwanted secondary
metabolite and phenolic compounds that can alter the purity of DNA or
RNA.

3. Place the collected sample on a zip lock containing ice or on a container

with silica gel inside to avoid degradation of the sample.

DEOXYRIBONUCLEIC ACID (DNA) EXTRACTION/ISOLATION

DEOXYRIBONUCLEIC ACID (DNA) is a substance within the cell

that carries the recipe for an organism and it's inherited by offspring from
parent. The DNA is located in the gene of an organism. Genomic DNA
comprises of all genes that are found in all living organisms. Before
undergoing any Downstream analyses such as gel electrophoresis,
Polymerase Chain Reaction (PCR), sequencing etc, the DNA of such
organism is first extracted or isolated. DNA extraction is carried out to
screen for genes of interest. Also, in modifying organisms genetically DNA
extraction is necessary.

DNA EXTRACTION PROTOCOLS

There are two major methods of extracting DNA, namely; Conventional

method and the Unconventional method. The Conventional method of
extracting DNA includes;

CTAB (Cetyltrimethylammoniumbromide) Protocol

SDS (Sodiumdodecylsulphate) Protocol

Phenol Protocol

Boiling Protocol

CTAB- PVP (Cetyltrimethylammoniumbromide- Polyvinylpyrrolidone)

The Unconventional method of extracting DNA is the Kit Protocol.

There are four stages involved in DNA extraction such as;

Lysis stage: This is subdivided into Mechanical and Chemical processes.

The mechanical process involves grinding the sample into powdery form
which helps to increase the surface area of the sample while the chemical
process is the application of extraction buffer to the extract.

Precipitation stage: This involves addition of non-polar solvent such as

Isopropanol to the supernatant where the DNA is present which result to
precipitation of the DNA from the supernatant after Centrifuging. The
reason a non-polar solvent is required for precipitation is because DNA is a
polar solvent that cannot be dissolved in non-polar solvent but will only be
precipitated.

Washing stage: The washing stage is also known as the purification stage.
It involves application of a non-polar solvent such as ethanol to the pellet
(DNA) which helps to eliminate contaminants. 70% ethanol is always
required in this stage.

Elution stage: It involves addition of a polar solvents such as sterile

distilled water, double distilled water, T.E buffer etc to the pellet (DNA). The
Polar solvent helps to solubilize the DNA, while protecting it from
degradation. This stage is done after the Washing stage.

CTAB PROTOCOL FOR GENOMIC DNA EXTRACTION OF SORGHUM

SEEDS

Aim: To extract or isolate the total nucleic acid on Sorghum seeds.

Plant Material: Fresh seeds were harvested from Sorghum bicolor plant
and kept in ice box to make them safe from degradation.

Equipment and Reagents needed: Mortar and pestle, Centrifuge,

Micropipette, Eppendorf tubes, Water bath, Vortex mixer, -40ºC freezer,
Liquid nitrogen, Extraction Buffer (T.E Buffer), Betamercaptoethanol, sterile
distilled water and RNase.

Procedures:

Preheat mortar and pestle at 60ºC for 15minutes using water bath.

Prepare extraction buffer using 10× CTAB, where for every 10ml of 10×
CTAB, add 80µl of Betamercaptoethanol.
Preheat the extraction buffer in the water bath.

Weigh 0.4mg of sorghum seeds using analytical weighing balance.

Grind the weighed seeds with sterile mortar and pestle using liquid nitrogen
to pulverize the hard substance of the seeds.

Add 800µl of extraction buffer in the mortar and pestle containing the
sample (grinded seeds).

Transfer the extract into 1.5ml Eppendorf tube and shake vigorously.

Incubate the extract at 60ºC for 10minutes.

Using Micropipette, add 60µl of Phenol, Chloroform and Isoamyl alcohol at

25:24:1 and vortex the mixture using vortex mixer.

Centrifuge mixture at 10000rpm for 10minutes.

Aliquot 450ml of the Supernatant into new 1.5ml Eppendorf tube.

Using Micropipette, add 450ml of ice cold Isopropanol into the new
Eppendorf tube containing the Supernatant and invert 4-5 times to allow
mixing.

Incubate the mixture for one hour at -20ºC.

Centrifuge at 10000rpm for 10minutes and carefully discard the

supernatant ensuring the pellet (DNA) is not disturbed.

Add 500µl of 70% ethanol to the pellet and centrifuge at 10000rpm for
5minutes. This process is done twice.

Decant the ethanol and air dry the DNA until it is completely dried.
 Suspend the pellet (DNA) in sterile distilled
water, then store in low temperature Freezer
at -40ºC for further downstream analyses.

Observations:

Centrifuging the mixtures at 10000rpm (revolution per minute) for

10minutes after addition of Phenol, Chloroform and Isoamyl alcohol, three
distinct layers were formed which are; The upper layer (Supernatant), the
middle layer (Denatured protein) and the lower layer (Organic compounds).

Addition of Isopropanol to the Supernatant, DNA was precipitated.

Conclusion: At the end of the experiment, a double stranded DNA was

formed.

Picture showing the three layers formed on addition of Phenol, Chloroform

and Isoamyl alcohol to the mixture and then centrifuging.
Loading extraction buffer into the eppendorf tube containing the sample.

DEOXYRIBONUCLEIC ACID (DNA) AND RIBONUCLEIC ACID (RNA)

QUANTITATION.

DNA/RNA quantitation is the determination of the concentration and

purity of the DNA/RNA. It involves the mechanism called
Spectrophotometer.

Spectrophotometer is the quantitative measurement of the amount of

light that passes (absorbs) through the solution of a solute in terms of
concentration at a particular wavelength.
 Quantitation of DNA/RNA is done using Nanodropspectrophotometer
and it follows the principle of Beer- Lambert which states that: "Absorbance
is directly proportional to concentration of the solution". It is mathematically
expressed as;

A∝cl

A=ϵcl

Where;

A= Absorbance

ϵ= Extinction coefficient

C= Concentration

l= Path length

PROCEDURES IN DNA QUANTITATION

Boot the system (Laptop/Desktop) and switch on the nanodrop

spectrophotometer.

Start the spec application and wait for it to load.

Micropipette 1µl of TE- EDTA (Tris- Ethylene diamine tetra acetic acid)
buffer or sterile distilled water depending on the polar solvent used in
eluting the DNA during DNA extraction, and drop it in the spec drop
surface, close the lid and click the blank button.

Micropipette out 50µl of DNA from each sample and place them on the
spec drop surface, close the lid, and click on the measure page from the
system.
 Results are shown at the end of the
process.

Picture showing the absorbance of Nucleic Acids(DNA/RNA) on spectrum.

NOTE:

DNA and RNA absorbs Ultraviolet light at 260nm while protein absorbs
Ultraviolet light at 280nm.

The ratio of 1.8-2.0 at A260/280 indicates a highly pure DNA sample.

The ratio of 2.0-2.1 at A260/280 indicates a highly pure RNA sample.

DNA samples with ratio lesser than 1.8(<1.8) indicates the interference of
phenolic compound or protein.

DNA samples with ratio greater than 2.0(>2.0) indicates the interference of
RNA.
 POLYMERASE CHAIN REACTION (PCR)

Polymerase Chain Reaction (PCR) is a molecular biology analysis for

rapidly amplifying DNA molecules in response to temperature changes. The
device used to carryout this molecular biology analysis is called
Thermocycler. PCR is commonly used in medical and biological research
laboratory for various work such as; identification of genetic fingerprints,
diagnosis of infectious diseases, cloning of genes, detection of hereditary
diseases, paternity testing etc.

REACTION MIXTURES IN PCR PROCESS

DNA Template: This contains the region of the DNA fragment that has been
extracted and ready for amplification.

Primers: These are short single- stranded nucleic acid used by living
organisms in the initiation of DNA synthesis. The primer functions in PCR
process by binding to the separated DNA template. There are two primers
used in PCR process, namely; The Forward Primer and the Reverse
Primer.

Deoxyribonucleotide triphosphates (DNTPs): It gives support to tiny DNA

structure.

Taq Polymerase: Taq (Thermus aquaticus) Polymerase is an enzyme that

aids the elongation or extension of the synthezied DNA structure.

PCR buffer: It is a reagent that helps stabilize the PCR reaction.

Mineral elements
Nuclease free water or sterile distilled water

Note: Master mix comprises of DNTPs, Taq Polymerase, Mineral elements

and PCR buffer.

THE REACTION PROCESS IN PCR

There are three major steps in PCR, which are; Denaturation, Annealing
and Elongation/Extension

Denaturation of DNA template: This involves the splitting of the double

stranded DNA structure at 92ºC- 95ºC.The solution contained in the tube is
heated to at least 92°C using a thermocycler. The heat breaks the
hydrogen bonds of the original DNA sample and separates the DNA into
single strands (this is termed denaturation of double-stranded DNA).

Annealing of the primers: It is the attachment of primers to the separated

DNA structure at 55ºC- 72ºC.The sample mixture is cooled to between
55°C to 72°C allowing the DNA primers and the DNA polymerase enzyme
to bind to the individual strands of DNA that were separated by the heat
(this is termed annealing of the primers). At this point, the nucleotides (A, T,
C, G) from the added mixture solution will pair with the individual separated
strands of DNA that resulted from the heating process.

Elongation/Extension of DNA strand: It is the addition of Taq Polymerase

that aids the elongation of the synthezied DNA structure. The temperature
is between 68ºC- 72ºC.Once joined together, they form a new
complementary strand of DNA (termed extension of the DNA). Thus, a new
duplicate double-stranded DNA molecule has been formed from each of the
single strands of the original sample molecule. The temperature cycles
from 95°C to 55ºC to 72°C. The cycle is then repeated about 35 to 40 times
using the thermal cycler which automatically repeats the heating and
cooling cycles of the process. Resulting DNA sequence is doubled each
time the heating/cooling cycle is conducted by the cycler. Thus, what
started out as a single short segment of DNA from one sample can be
amplified to form millions of copies after 35 doubling cycles.

PCR PROCEDURE

Arrange 1.5ml Eppendorf tubes on a rack and place them on ice.

Remove the nuclei acid stock solution in tube from the genebank (-40ºC
freezer), and place it on ice.

Micropipette 1µl each of the nuclei acid stock solution i.e the DNA template
into the new 1.5ml Eppendorf tubes.

Add 1µl each of both forward and reverse primers to the tubes containing
the DNA template.

Micropipette 8µl each of the master mix(DNTPs, Taq Polymerase, mineral

elements and PCR buffer) and place on the tubes.

Add 6µl each of sterile distilled water to the tubes containing the mixture.

Switch on the thermocycler and place the tubes containing the mixture
inside it, then set the needed temperature for the reaction.

GEL ELECTROPHORESIS

Gel Electrophoresis is a technique that involves the determination of

fragment of DNA sizes, separating them in a gel placed in an electric
current. There are two types of gel electrophoresis, namely; Agarose gel
electrophoresis and Polyacrylamide gel electrophoresis.

AGAROSE GEL ELECTROPHORESIS: It is a technique used to separate

large DNA/RNA molecules, large protein and protein complexes. It is
arranged in a horizontal form.

POLYACRYLAMIDE GEL ELECTROPHORESIS: This is a type of gel

electrophoresis used to separate smaller DNA/RNA molecules and large
protein. It is arranged in a vertical form.

PREPARATION OF 2% AGAROSE GEL

Equipment and Reagents needed: Agarose powder, 1× TBE buffer,

Nucleic acids stain (Ethidium bromide/Cyber green), sensitive weighing
balance, microwave, measuring cylinder and beaker.

Procedures:

Weigh 1.0g of agarose powder accurately using an analytical weighing

balance.

Dissolve the agarose powder in 50ml of 1×TBE buffer.

Boil the solution for 1-2 minutes using a microwave.

Allow the solution to cool at room temperature and add nucleic acid stain to
the solution.

Pour the solution in a gel cast tray containing the gel combs and allow it to
solidify.

Carefully remove the combs without splitting the gel, especially around the
wells.
 Prepare the gel electrophoresis
tank and fill with 1×TBE buffer

Transfer the gel into the gel

electrophoresis tank containing
1×TBE buffer making sure the
well is facing the negative pole
of the tank. This is done so that
when current passes through
the well, the DNA will migrate to
the positive pole.

The gel is now ready to be loaded with samples.

 Loading nucleic acid stain on
agarose solution.

Picture showing Agarose gel on a gel tray

STEPS IN LOADING AND RUNNING AGAROSE GEL

Load the loading dye on the first and last well of the gel

Load the amplicon (amplified DNA) on the other wells aside the first and
last well of the gel.

Connect the wire to the two poles (positive and negative) of the gel
electrophoresis tank and connect it to a power source.
Allow the DNA to migrate to the positive pole before transferring the gel to a
Ultraviolet documentation to view the DNA.

Note:

Microwave is used to boil the solution because it is faster and doesn't

evaporate the solution which can affect the gel concentration.

The Nucleic acid stain helps the DNA to be visualized under the UV
documentation.

TISSUE CULTURE LABORATORY

This section is under Biotechnology department and mostly supports

the mandate of the Company. Tissue culture unit engaged mainly in in-vitro
conservation, regeneration and multiplication of both agronomic and tree
crop species, on a nutritional medium under an aseptic environmental
condition.

The Tissue culture laboratory houses some certain important sections

which are: Growth room/Transfer section, Media preparatory room,
washing and storage room, screen house, post flask and the TIBS
(Temporary Immersion Bioreactor System).

Plant Tissue Culture: Plant Tissue Culture is the invitro propagation of

plant cells, group of cells, tissues, and organs in an artificial culture medium
under aseptic condition.

Advantages of Plant Tissue Culture

The new plants produced are disease free.

The plantlets are obtained in a very short time with a small amount of plant
tissue.

The plants can be grown throughout the year, irrespective of the season.

A large space is not required to grow plants.

The production of new varieties in the market place speeds up.

Applications of Plant Tissue Culture

Mass propagation of plant.

Crop improvement.

Conservation of genetic resources.

Production of secondary metabolite.

Invitro breeding.

It can also be applied in genetic engineering.

Biosafety precautions in Tissue Culture Laboratory

Always put on your lab coat whenever you're in the laboratory.

Do not eat, drink or smoke in the laboratory.

Switch off your handset to avoid distraction.

Always put on your hand gloves and do not use gloved hands to pick
personal materials.

Handle all bottled reagents with care to avoid spillage and do not inhale
reagents.
 Report every spill or accident to the lab scientist.

Switch every electrical equipment after use.

EQUIPMENT IN TISSUE CULTURE

LABORATORY

Autoclave: This is an apparatus used to sterilize media, instruments and

glasswares. Autoclave sterilizes media at 121ºC for 15 minutes and
sterilizes glasswares or instruments at 121ºC for 30 minutes.

An Autoclave
 Water Distiller: It is used to purify water by
eliminating contaminants, including chemicals,
heavy metals, microorganisms and sediments
present in the water.

Water Distiller

Deionizer: It is an equipment used to remove ions and minerals by

synthetic ion exchange resins, thereby making the water pure and ion free.
 A Deionizer

pH meter: It is used to check for the degree of acidity or alkalinity of a

solution.
 A pH meter

Microwave: It is used for boiling prepared media.

A Microwave

Culture Vessels: These are containers used to store media and inoculated
plant.
 Culture Vessel

Analytical weighing balance: It is an equipment used to weigh reagents

needed for media preparation.

Analytical weighing balance

Drying Oven: It is an equipment used to remove moisture from the oven
chamber so as to dry the equipment as quickly as possible. It is also used
in sterilizing instruments. It sterilizes instruments at 160ºC for one hour.

A Drying Oven

STEPS IN TISSUE CULTURE

The steps in tissue culture include; Glassware preparation, media

preparation, collection of explant, disinfection of explant, inoculation of
explant, incubation of culture, sub- culturing and transplanting of
regenerated plant.

GLASSWARE PREPARATION
 Glassware such as Test tubes, petri dishes, Beaker, Pipette, Reagent
bottle, Conical flask etc. are prepared by washing and sterilizing them.

Washing of glassware involves cleaning glassware with soap, jig and water.
This is done to eliminate dirt from the glassware thereby making them
clean for use.

Sterilization of glassware can be done using Autoclave (Moist heat

sterilization) and Drying Oven (Dry heat sterilization). Autoclave sterilizes
glassware at 121ºC for 30 minutes while Drying Oven sterilizes glassware
at 160ºC for one hour or 170ºC for 30 minutes.

MEDIA PREPARATION

Media are solid or liquid substrata on which, or in which, cells or

organ explants can be made to grow. We have various types of medium
such as Murashige and Skoog (MS) medium, Linsmaier and Skoog (LS)
medium, Gamborg (B5) medium, Nitsch and Nitsch (NN) medium, White’s
Medium etc.

Procedures in Preparing 750ml of Yam Proliferation media

Measure approximately 500ml of deionized water into a 1000ml beaker.

While stirring the water using magnetic stirrer, add 50ml of macronutrient
solution (stock 1).

Continue stirring the mixture while adding 5ml of micronutrient solution

(stock 2)

Add EDTA and FeSO4 of the required gram. (stock 3).

NOTE: EDTA must be added before FeSO4.

Then add vitamins (stock 4) and sucrose as the carbon source.
 Growth hormones such as Auxin and
Cytokinin are added in the required
concentration.

Add additional deionized water to bring the

medium to almost the final volume (750ml)

While stirring, adjust medium to desired pH

(5.8) using NaOH, HCL or KOH.

Then add a gelling agent (Agar) and stir the medium using magnetic stirrer.

Using microwave, boil the medium for 1- 2minutes.

Dispense the medium into the culture vessels before autoclaving.

Sterilize the medium using an autoclave at 1kg/cm 2 (15psi), 1210C, for 15minutes
( this depend on the quantity of the media)

Allow to cool prior to use.

Weighing the required Agar for media preparation

 EXPLANT COLLECTIONS

Collection of vegetative plant materials (germplasm) could be from

the field, Screen house or growth room. Examples include; Cassava nodal
cutting, Costus nodal cutting, yam vine cutting etc.

Procedure in Collecting Explant

Using cleaned and sterilized collections bottles, place transport medium in

collection bottles.

Use scalpel to excise ex-plant from the mother plant.

Place labeling tape on collection bottles and label properly for easy
identification.

Disinfection procedure

Wash ex-plant thoroughly under running water and little liquid soap to
eliminate surface dirt.

Do final rinsing with distilled water.

Add 70% ethanol for 5 minutes and decant.

Note:

There are two types of disinfection, namely; Single and Double

Disinfection

Single disinfection

Use 10%v/v sodium hypoclorite (NaHPO4) for 20 minutes and decant.

Rinse thrice using sterile water

Double disinfection
Use 10%v/v sodium hypoclorite (NaHPO4) for 20 minutes and decant

Rinse once and decant

Add 5%v/v sodium hypochlorite (NaHPO4) for 10 minutes and decant.

Rinse thrice with sterile water

Leave in the third rinse till ready for use.

Procedure in Inoculating Explant

Switch on the flowhood and sterilize the surface with 70% ethanol.

Spray hands with 70% ethanol.

Arrange neatly all culture materials inside the flowhood and sterilize them
with 70% ethanol.

Lit the spirit lamp with matches.

Excise explants using sterilized petri dish, forcep and blade.

Inoculate the excised explant into the medium.

Seal and label with date and name of the plant.

Then place the culture in the growth room ensuring the necessary
conditions for growth such as light is present in the growth room and.

INCUBATION OF CULTURE

After inoculation, the cultures are incubated in culture room or in a

BOD incubator at 25±2°C temperature. For certain plant or for some
particular culture type below or above 25°C is needed.
 Some tissues grow well under low light condition (approx. 1000 lux),
for regeneration light and dark periods are needed, and for regenerated
plantlet well lighted (approx. 3000 lux) condition and 16h light with 8h dark
period is needed. The illumination in the culture room is provided by cool
white fluorescent light placed approx. 18″ above the culture racks.

Low humidity causes the quick desiccation of culture medium and

high humidity is favourable for contamination of culture medium. Specific
relative humidity (20-98%) is to be maintained in the culture room and
uniform air circulation is to be done properly.

For cell suspension culture agitation and aeration is secured by the

use of shaker systems; open platform orbital shakers or the orbital
incubators fitted with fluorescent lights to provide different day/night
regimes.

SUB- CULTURING OF EXPLANT

The growth and development of tissues cultured in-vitro are generally

monitored by observing the cultures at regular intervals in the culture room
or incubators.

Based on the observations either with hand-lens or with the aid of

simple microscope under aseptic conditions, the explants may be required
to transfer to new media (freshly prepared) or with new ingredients or
hormone composition depending on the state of growth of cell or tissue.

The same precautions and full aseptic conditions are maintained during the
transfer process also. The delaying of this process may lead to inhibition of
proper development of tissues and also delaying the regeneration of
plantlets.
 In case of suspension culture the change of media or fresh
inoculation at quick intervals is needed and also for callus culture the sub-
culturing of the callus tissue is needed to get the callus tissue in dividing
conditions.

TRANSPLANTATION OF REGENERATED PLANT

Plants regenerated from in-vitro tissue culture are transplanted to soil

in pots. Prior to transfer to pots the acclimatization of these regenerated
plants are needed. The plants at this time develop adequate root systems
and cuticular leaf surface structure so that it can withstand the field
environmental condition.

The process of acclimatization needs the humid chamber and a slow

process to make the plantlet habituated from high humid condition to
normal atmospheric humidity. The greenhouse or the growth chamber
should have artificial light system also which includes a mixture of
fluorescent and incandescent lamps designed to provide balanced
wavelengths of light for plant growth and photosynthesis.

The greenhouse facilities are needed for winter crops and summer
crops differently for maintenance of proper temperature, required, air
circulation and the relative humidity. The potted plants are grown in field for
further observation, flowering and normal seed setting to get the next
progeny.

ACCLIMITIZATION/ POSTFLASK MANAGEMENT

Postflask Management
 Postflask management refers to the acclimatization, hardening, or
weaning of tissue culture plantlets after they are removed from culture
containers. It is an important step in the transfer of tissue culture plantlets
to field conditions. This is an intermediate stage (transition) between the
culture containers and the field.

The main factors affecting the survival of these plantlets after they are
transplanted from culture containers to the growing medium are:

The moisture in the medium

Relative humidity

Light intensity

Temperature

The main differences between tissure culture plantlets and field/

greenhouse grown plants are in the structure of the plant and the growing
environment.

The net photosynthesis and carbon metabolism of tissue culture

plantlets decreases after plantlets are transplanted to the soil. Full recovery
is usually observed one to two weeks after transplanting in-vitro plantlet in
a medium that provides optimum nutrient requirement and aseptic
conditions. Acclimatization of micro propagated plantlets can start from in-
vitro by gradual reduction of relative humidity inside the culture container
and exposure of culture to higher light intensity.

However, it is more common that plantlets are transplanted to high

humidity conditions in an enclosed area (chamber e.g. polyethylene/tents)
under the shade. The growing medium and chamber with which Cultures
and plantlets are transplanted is very important for good survival. Inhibition
or dramatic shift in pH in a medium can adversely affect root growth .And
this transplanting success sufficient porosity of the growing medium to
allow adequate drainage and aeration is also important for good
establishment. The application of mild organic and inorganic fertilizers of
different concentrations prior or after potting/field establishment could also
improve plants vigor after acclimatization.

Post flask management technique can be described under six stages:

Plantlets preparation: Plantlets to be acclimatized must have well defined

shoots and roots. It must be free from any forms of mechanical damage
and contaminations.

Safe handling of plantlets: Tissue culture plantlets are very tender, fragile
and sensitive to shock. Therefore optimal care is required in handling it, to
prevent damage prior transplanting.

Preparation of transplanting kits: The materials /medium used for

transplanting must be sterile to prevent contaminations, medium must be in
the right proportions.

Transplanting: Plantlets are labelled with plastic tags prior to its removal
from the culture vials. The plantlets removed are washed in a container of
water in other to wash off the media on the roots to discourage microbial
growth. The roots are immersed completely and covered with sterile soil
medium then kept inside a white polythene bag, watered, tied properly and
then kept inside acclimatization chamber to avoid wilting due to possible
evapo-transpiration in the outside environment.

Releasing of humidity chamber: The TC plantlets are kept in the

acclimatization chamber for an average of one to two weeks before
chamber is opened to release the humidity. The period at which plantlets
are retained in the chamber depends largely on the performance of the
plantlets.

Potting/Field establishment: When plantlets have been fully acclimatized,

they undergo a stage of weaning called potting” whereby soil compositions
are prepared and potted in black polythene bags prior potting of plantlets.
And when the plantlet has fully developed to seedlings it’s then transferred
to the field for establishment.

The three main postflask activities are; Acclimatization, Screening

(Screenhouse) and the Field.

ACCLIMATIZATION

Acclimatization, the first postflask activities involves the weaning of in-

vitro established plantlets from the laboratory to the acclimatization
chamber. This activity is carried out immediately after the plantlets have
been well nurtured in-vitro. It serves as a pathway between the in-vitro
propagation and the external exposure to the atmosphere. Note that certain
things are usually looked into during this process or stage. For instance,
the soil used must have the following components;

Coconut fibre (grinded): It helps in softening the rigidity of the top soil since
the plantlet roots are pseudo.
Stone dust or river sand: It serves as a permeable membrane for the
medium. This is because stone dust or river sands are porous in nature.

Top soil: It provides the plantlets rigidity or firmness when present in the
acclimatization medium.

AIM IN ACCLIMATIZATING PLANTLETS

Acclimatization is aimed at weaning about 100% of the fully established

plantlets from the in vitro to the ex-vitro.

To mass produce plants that is disease free.

Acclimatization is an intermediary stage between the in-vitro and the ex-

vitro nurturing of plants. It basically serves as an adaptation system or
ground for fully established weaned in-vitro plantlets.

PROCEDURE IN ACCLIMATIZING PLANTLETS

Collect the already established plantlets from the growth room.

Remove the plantlets and rinse them properly in water.

Sterilize the soil at 250°C for an hour.

Open up some polythene plant bags and fill them up with the specially
prepared sterile soil that has been oven dried for about an hour at 250°C.

Inoculate the plantlets into the sterile soil filled plant bags and place them in
an acclimatization tray.

Spray the plantlets with a distilled water filled post flask.

Wrap in transparent polythene and hang the plantlets in the acclimatization

chamber.
Place them in the acclimatization chamber for 7 days before puncturing the
polythene bags weekly for easy air passage, watering and acclimatization.

The plantlets stay for twenty one days in the chamber before they are
finally transported to the screen house.

The factors affecting plantlets are, extreme weather condition, high degree
of contamination from the in-vitro culturing etc.

USES OF ACCLIMATIZATION

It helps in providing 100% survival rate in plantlets.

It aids in attaining high relative humidity condition in ex vitro condition.

It helps in hardening of the plant tissues (xylem and phloem) i.e. it helps in
re-establishing the plantlets with rigid and well established tissues.

It helps produce true-to-type plantlets (this is achieved by totipotency and

plasticity).
 Picture showing an Acclimatization Chamber

SCREENING (SCREEN HOUSE)

This is the next postflask activity after acclimatization. The plantlets

are transported to the screenhouse for continuous growth and adaptation to
the external environment. In the screen house, the plantlets are placed in a
more comfortable soil filled, durable and water containing polythene bags.
The plantlets are watered daily, until they are fully elongated and well
established for transplanting finally to the field.

FIELD

This is the final stage in tissue culture processes because the

plantlets are planted in the field after screening has been carried out.
Plants are then cultivated after all these several stages have been
administered from the laboratory to the field where the plants are finally
catered and cared for.
Advantages of Post flask Management to Tissue culture Plantlets.

It creates high level of humidity for the survival of in vitro plantlets.

It is a transition stage that enhances gradual adaptation of plantlets from in

vitro to ex vitro condition.

It prevents plantlets from abiotic stress (low humidity, direct sunlight, wind,
heavy rain drops).

It prevents dehydration of plantlets in an in vitro condition.

It provides an absolute establishment of 100% survival of the plantlets for

field establishment.

FIELD GENEBANK

Field gene-bank is also a section under Plant Genetic Resources

(PGR). The field gene-bank unit is responsible for the management of all
Recalcitrant plant species [seeds whose moisture content cannot be
reduced to an appreciable level without losing their viability e.g Kigelia
africana (sausage tree), Spongias mombin (Hog plum), Pluckenetia
conophora (Africa walnut) etc].

The focus of the unit is to collect and conserve indigenous recalcitrant

plants that are threatened with extinction, endangered endemic plants,
economic tree crops, underutilized and wild plant species and varieties of
crop species such as root and tubers, fruit tree crops and other food crops
with recalcitrant seed behaviour. Likewise, the unit also manages safety
duplicates of newly registered and released varieties of such related crop
species.
 There are two methods by which this recalcitrant plant can be
conserved, namely; LIFE CONSERVATION AND HERBARIUM METHOD
OF PRESERVATION.

1. Life Conservation: This requires the following procedure

exploration/collection of valuable genetic resources ranging from vegetative
to reproductive portion of the plant after which they have been raised at the
nursery section via a best suitable method either vegetative or reproductive
till the plant attain the stage of maturity before they will be finally
transplanted and established permanently at the field.

Plants are conserved on the field in two ways these are: Ex-situ and In-
situ method of conservation.

EX-SITU: This is the conservation of plants outside their natural habitats.

IN-SITU: This is the conservation of the component species in their natural

surrounding where they have developed their distinctive properties.

2. Herbarium method of preservation: Herbarium is a plant museum or a

plant library where meaningful portion of plant are collected assembled and
processed and dried for sustainable utilization. This section keeps,
preserves (plants part) and conserves plant specimens that will help in
accurate identification and description of plants and their locations and bio-
geographical distributions within and outside the country.

HERBARIUM FLOW CHART