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

ByBy

Arinaitwe A.B, Namugga. P, Tibanyedera. D and ImeldaArinaitwe A.B, Namugga. P, Tibanyedera. D and Imelda

Speke Resort Munyonyo, KampalaSpeke Resort Munyonyo, Kampala

1/11/20121/11/2012

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, Rukungiriand 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

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.

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, Mwengebigere, 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.

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

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

Tissue culture of potato for production of quality seed potato

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)

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.

What is plant tissue culture? Cont’d

1

9

8

6, 7

5

4

3

2

Setting of the plant tissue culture laboratory

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)

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 KachwekanoStation

• The low yield accounted for by constraints to potato productionwhich 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) andinadequate availability clean seed,

Key potato production challenges

Potato leaf roll virus

Bacterial wilt

Late blight

Potato virus Y and Potato virus X

Inadequate seed potato

• 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

Why use biotechnology in potato contn’d

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)

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.

SEED PRODUCTION CAPACITYSEED PRODUCTION CAPACITY

Tissue culture laboratoryTissue culture laboratory

Potato plantlets growing in Potato plantlets growing in the growth room at KAZARDIthe growth room at KAZARDI

••Current capacity is about Current capacity is about 10,000 plantlets10,000 plantlets

••Seven (7) varieties have been Seven (7) varieties have been successfully propagatedsuccessfully propagated

Potato Tissue culture plantlets in the Tissue culture labaratory at

Kachwekano

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

Potato plantlets growing in Aeroponic facility at Kachwekano

Potato plantlets growing in Aeroponic facility at Kachwekano

Minituber production at Kachwekano In Aeroponic facility

AEROPONICS SCREEN HOUSEAEROPONICS SCREEN HOUSE

Capacity of 1440 plantlets per

season

•Four varieties have been

successfully tested

•Each plant averagely gives 40

minitubers

Potato plant with Potato plant with minitubersminitubers growing in the growing in the aeroponicsaeroponics screen house at KAZARDIscreen house at KAZARDI

Minituber production in soil

SEED MULTIPLICATIONSEED MULTIPLICATION

•• From the screen house,From the screen house,

•• Two field multiplication cycles to generate Two field multiplication cycles to generate basic seedbasic seed

•• Commercial seed producers (basic seed)Commercial seed producers (basic seed)

•• Technical backstopping Technical backstopping

BASIC SEED PRODUCTION AT KACHWEKANOBASIC SEED PRODUCTION AT KACHWEKANO

Varieties Available Varieties Available

•• NakpotNakpot 1 1

•• NakpotNakpot 5 5

•• KachpotKachpot 11

•• KachpotKachpot 22

•• Victoria (.20)Victoria (.20)

•• RutukuRutuku (Uganda 11) (Uganda 11)

•• SangemaSangema

•• CruzaCruza

� Genetic engineering: This is tThis is the process of manipulating and he process of manipulating and transferring instructions carried by genes from one cell to anottransferring instructions carried by genes from one cell to anotherher

Genetic engineering in potato targeting Genetic engineering in potato targeting transfer of bacterial wilt resistance genes transfer of bacterial wilt resistance genes

Why do scientists carry out plant genetic engineering?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

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

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)

Elements of plant transformation

1. Biological methods

Agrobacterium -mediated transformation

2. Direct methods

-Protoplast microinjection

-Particle bombardment

Method of delivery of DNA into the cell

Considerations of Plant Genetic Engineering

Choice of Plant part to be used (explant)

This could be Embryogenic Cell Suspension (ECSs), callus or internode

ECSs

Selected explant or tissue must be regenerable before transformation

�After transformation process, cells must be regenerated into fertile plants

Potato Callus

Internodes

Cloned DNA (gene) to be introduced

Marker

geneTerminator Terminator gusA RL

Genetic transformation in Potatoes� Two transformation systems are commonly used

Particle mediated gene transfer

Histochemical GUS assay

Selection and regeneration

Agrobacterium gene transfer system

Infection

Co-cultivation

Selection and regeneration

4-5 days3-4 days

Trasformation and regeneration process in

Potatoes

Embryogenic CallusSelection of transgenic on

kanamycine anti-biotic Gene transfer using

agrobacterium

Infect the callus with

Agrobacterium

strain(EHA105, AGL1)

containing a suitable

vector

Transgenic linesinternode

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

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

Thank you