Presentation by Abel Arinaitwe, KAZARDI, Uganda Delivered at the B4FA Media Dialogue Workshop, Kampala, Uganda - November 2012 www.b4fa.org

1. Use of biotechnological techniques in production of
quality seed potato in Uganda
A case study of Kachwekano Zonal Agricultural Research
and Development Institute in Kabale
By
Arinaitwe A.B, Namugga. P, Tibanyedera. D and Imelda
Speke Resort Munyonyo, Kampala
1/11/2012

2. Mandate of
Kachwekano ZARDI
• Kachwekano Zonal Agricultural Research and Development
Institute(KAZARDI) is one of the NARO’s Public Research institutes and is
located in Kabale district in south western Uganda.
• .It is mandated to spearhead agricultural research and development for
increasing agricultural productivity in Uganda's South western highland
agro ecological Zone (SWHAEZ) that comprise of Kabale, Kisoro, Rukungiri
and Kanungu districts
• Developing and availing improved technologies, practices and tools for
use by producers, processors and traders in order to improve agricultural
productivity and agro- enterprise
• Production of quality planting and stocking materials of potato, apple,
pear, sorghum, wheat , barley crops and goats among the livestock

3. What is Biotechnology?
• Biotechnology is application of techniques
using biological systems, living organisms or
derivatives from them to make or modify
products or processes for use by humankind.

4. Techniques in biotechnology
• First generation biotechnological techniques included
fermentation, plant and animal breeding that have been used
to produce products and improve certain traits.
Biotechnology is not new and has been used to produce food
and drink (yoghurt, cheese, beer - Ajon, Muramba, Mwenge
bigere, wines, yeast, etc).
• The second generation of Biotechnology includes, use of cell
culture or tissue culture to yield chemical products and
growing tissues and plants (clonal plants).
• Modern Biotechnology involves application of recombinant
DNA technology by introducing nucleic acid material (DNA)
containing the genetic code (genes) in to other cells naturally
or artificially.

5. Techniques used at Kachwekano
ZARDI
• Kachwekano ZARDI is using;
• Tissue culture- for Rapid microprogation to produce
enough planting materials (seed potato) (2006)
• Recombinant DNA technology- for Gene transfer to
combat most production constraints- (since 2012) in
collaboration with NARL-Kawanda
• Immunology- For Disease diagnosis using ELISA based
methods , virus elimination and bacterial wilt detection

6. What is plant tissue culture?
•
The technique of growing plant cells, tissues and organs in an
artificial prepared static or liquid nutrient medium, under
aseptic conditions (Clean conditions).
•
The technique is often referred to as in vitro culture. The term in
vitro literally refers to “in glass” or “in test tube” or “in bottle”.
•
A piece of a plant, which can be anything from a piece of stem,
root, leaf, or bud to a single cell, is cut and placed in that tiniest
a test tube and used to develop new plants. In this environment
free from microorganisms and in the presence of a balanced diet
of chemicals, the explant, can produce plantlets that in turn, are
able to multiply indefinitely

7. Tissue culture of potato for production of quality seed potato

8. What is plant tissue culture? Cont’d
Plant tissue culture techniques are generally characterized by;
1.
Work being done on a small-scale
2.
Environmental conditions are optimized with regard to physical, nutritional and
hormonal factors of the plants
3.
All microorganisms (fungi, bacteria and viruses), as well as other pests of higher
plants are excluded i.e. it is performed under sterile conditions
4.
The normal pattern of plant development often breaks down, and an isolated tissue
can rise to calli or can develop in many unusual ways (e.g. organ formation, somatic
embryogenesis)

9. What is plant tissue culture? Cont’d
The stages in plant tissue culture;
1.
Obtain the explant (A piece of plant tissue taken out from original site of plant and
transferred to an artificial tissue media for growth or maintenance)
2.
Clean the explant
3.
Media preparation
4.
Media sterilization
5.
Multiplication of explant
6.
Rooting
7.
Shooting
8.
Acclimatization/Hardening off
9.
Transplanting to container/field
•
These stages can overlap in certain cases, and the requirements of each stage vary
widely from plant to plant.

10. What is plant tissue culture? Cont’d
2
1
5
3
4
6, 7
9
8

11. Setting of the plant tissue culture laboratory

12. Culture media
Components/ Requirements of culture media
•
Inorganic salts (nitrate, potassium and ammonium)
•
Plant growth regulators (Auxins, Cytokinins, Gibberellins, Abscisic acid)
•
Vitamins (Vit B1, Vit B2, Vit B6, B12, Vit C)
•
Energy source (Sugar or sucrose)
•
Proteins (Amino acids and amines)
•
Antibiotics (for controlling micro-organisims like bacteria and fungi)
•
Gelling agent (something to hold the components of the media)

13. Why use biotechnology in potato
• Uganda stands at 33 million people and is projected to be 53
milllions by 2025 and 94M by 20 50. With this increase in population,
there is likely to be both food and nutritional insecurity.
• Potato, (Solanum tuberosum L.,) is grown by 300,000 small holder
households that produce a total of 650,000 tons on 93,000 hectares
(FAOSTAT, 2007).
• Potato yields at farmers’ fields have stagnated at about 7 tons per
hectare compared to the 18 - 25 tons per hectare at Kachwekano
Station
• The low yield accounted for by constraints to potato production
which include; late blight (LB) and bacterial wilt (BW) viruses Potato
leaf roll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato
virus S (PVS), Potato virus A (PVA) and Potato virus M (PVM) and
inadequate availability clean seed,

14. Key potato production challenges
Bacterial wilt
Potato virus Y and Potato virus X
Potato leaf roll virus
Late blight
Inadequate seed potato

15. Why use biotechnology in potato contn’d
•
95% of farmers have traditionally relay on their own local Seed potato supply that is
obtained by keeping the small-sized (non marketable for table consumption) from the
previous crop harvest because of inadequate clean planting materials.
•
The quality or planting value of home saved seed potato in general is very poor and in
most cases it is infected with bacterial wilt and viral diseases.
•
Conventional breeding for bacterial wilt resistance in potato has been very challenging
with limited success due to a very narrow genetic and variable polyploidy levels
(different chromosomes numbers (n-6n ) in potato male sterility.
•
Therefore; Uganda undeniably needs clean planting materials for farmers , bacterial wilt
resistant potato varieties , more food, energy and a higher standard of living, for its
people.
•
Biotech has the potential to address the above challenges, and presents a feasible
solution to production of clean planting material in short time, increase crop yields and
improve livelihoods of many farmers in Uganda
It was on this premise that this KAZARDI started use of biotechnological techniques to
address the challenges limiting the production of quality seed
•

16. Use of Tissue culture to address the challenge of quality seed
potato at Kachwekano
Objectives of establishing Tissue culture Lab at Kachwekano
• To multiply plant tissues, organs under sterile conditions in the
absence of pathogens like( fungi, viruses, bacteria etc.) using
Tissue culture techiniques
• To Produce high quality potato planting material free from
diseases
• Rapidly multiply high quality potato plants to generate enough
seed for the farmers
• Preserve germplasm (Potato varieties) and overcome genetic
erosion (Loss)

17. Why tissue culture is possible in plants
•
The phenomenon of cell totipotence is a cornerstone to the tissue culture
technique. Living plant cells undergo cell division to give an entire plant of
the same genetic constitution as the mother plant.
•
This indicates that each plant cell contains all the necessary information for
regeneration of a new plant.
•
The multiplication of plants in vitro does not establish any new processes
within the plants. Tissue culture simply directs and assists the natural
potential within the plant to put forth new growth and to multiply in a
highly efficient and predictable way.

18. SEED PRODUCTION CAPACITY
Tissue culture laboratory
Potato plantlets growing in
the growth room at KAZARDI
•Current capacity
10,000 plantlets
is
about
•Seven (7) varieties have been
successfully propagated

19. Potato Tissue culture plantlets in the Tissue culture labaratory at
Kachwekano

20. Production of potato minitubers
• From the Tissue culture Laboratory, the
plantlets are planted in the soil or Aeroponic
facility where they are grown suspended in air
(Aeroponic) for production of minitubers
(Nuclear seed) (First seed generation)
• From this nuclear seed, pre-basic and basic
seed will be generated

21. Potato plantlets growing in Aeroponic facility at Kachwekano

22. Potato plantlets growing in Aeroponic facility at Kachwekano

23. Minituber production at Kachwekano In Aeroponic facility

24. AEROPONICS SCREEN HOUSE
Capacity of 1440 plantlets per
season
•Four varieties have been
successfully tested
•Each plant averagely gives 40
minitubers
Potato plant with minitubers growing
aeroponics screen house at KAZARDI
in
the

25. Minituber production in soil

26. SEED MULTIPLICATION
• From the screen house,
• Two field multiplication cycles to generate
basic seed
• Commercial seed producers (basic seed)
• Technical backstopping

27. BASIC SEED PRODUCTION AT KACHWEKANO

28. Varieties Available
•
•
•
•
•
•
•
•
Nakpot 1
Nakpot 5
Kachpot 1
Kachpot 2
Victoria (.20)
Rutuku (Uganda 11)
Sangema
Cruza

29. Genetic engineering in potato targeting
transfer of bacterial wilt resistance genes
Genetic engineering: This is the process of manipulating and
transferring instructions carried by genes from one cell to another
Why do scientists carry out plant genetic engineering?
A. Addressing concerns of production constraints in plants like
resistance to bacterial wilt disease and other diseases.
B. Testing function of genes or parts of genes
A. B. Modifying Expression of Endogenous Genes
- Turning genes off
- Increasing Expression
- Modifying Expression
C. Moving genes from other organisms

31. Characteristics a gene transfer system
DNA delivery systems must be
- simple, efficient and preferably inexpensive
One’s system of choice depends on
- the target tissue,
- its regeneration system,
- the position or affiliation of the researcher

32. Elements of plant transformation
A.
Cell culture and plant regeneration system
B.
Cloned DNA to be introduced (gene of intrest) with these parts below
1. Selectable marker gene
2. Promoter (constitutive or inducible),
3. Coding region
C.
Method of delivery of DNA into the cell (Transfer to another
organism)
D.
Proof of transformation (molecular biology analyses – PCR, RT-PCR,
Southern and Northern blot assays)

33. Method of delivery of DNA into the cell
1. Biological methods
Agrobacterium -mediated transformation
2. Direct methods
-Protoplast microinjection
-Particle bombardment

34. Considerations of Plant Genetic Engineering
Choice of Plant part to be used (explant)
This could be Embryogenic Cell Suspension (ECSs), callus or
internode
Internodes
ECSs
Potato Callus
Selected explant or tissue must be regenerable before transformation
After transformation process, cells must be regenerated into fertile plants

35. Cloned DNA (gene) to be introduced
L
Marker
gene
Terminator
Terminator
gusA
R

36. Genetic transformation in Potatoes
Two transformation systems are commonly used
Particle mediated gene transfer
Agrobacterium gene transfer system
Infection
3-4 days
4-5 days
Co-cultivation
Histochemical GUS assay
Selection and regeneration
Selection and regeneration

37. Trasformation and regeneration process in
Potatoes
internode
Embryogenic Callus
Gene transfer using
agrobacterium
Infect the callus with
Agrobacterium
strain(EHA105, AGL1)
containing a suitable
vector
Selection of transgenic on
kanamycine anti-biotic
Transgenic lines

38. Planned future work
• Continue with regeneration and transformation of
potatoes with bacterial wilt resistance genes
• Evaluating the potato varieties generated with genetic
engineering for resistance to bacterial wilt
• Putting together different genes (gene stacking) for
resistance for bacterial wilt in to potato to avoid
resistance breakdown
• Looking for drought tolerant genes and putting them in
potato to address the issues of climate change

39. Conclusion:
• In Uganda, the Government has embraced
biotechnology as a strategy to enhance
agricultural productivity and industrialization,
improve health care delivery and preserve the
environment.
• So lets join Hands and feed the future
generation

40. Thank you