The document discusses biotechnological innovations being developed at the Coffee Research Centre in Uganda to multiply coffee varieties resistant to coffee wilt disease for farmers. Some key points: 1) Tissue culture techniques are being used to rapidly produce millions of coffee seedlings from a single leaf, offering a major improvement over traditional propagation methods. 2) Embryogenic cultures are induced from leaf explants and grown in bioreactors to generate somatic embryos, which are conditioned, weaned, and grown to planting size. 3) Several new coffee wilt disease resistant varieties have been developed and are being multiplied using these techniques to provide farmers with disease-resistant materials to improve yields.

1. DEVELOPING BIOTECHNOLOGICAL INNOVATIONS
TO ENHANCE MULTIPLICATION OF COFFEE WILT
DISEASE RESISTANT MATERIALS FOR FARMERS
Africano Kangire
Israel Sebugenyi, Pascal Musoli and Naboth Edongot
COFFEE RESEARCH CENTRE, UGANDA
P.O.Box 185, Mukono-Kituza
Email:afrikangire@gmail.com
Presentation at Biosciences For Farming in Africa, Kampala 1st Nov., 2012

2. COFFEE RESEARCH CENTRE, AT KITUZA,
MUKONO.

3. COFFEE AND ITS IMPORTANCE IN UGANDA
• Over 8 million people derive their livelihood directly from
coffee (involvement at different levels)
• Uganda produces both Robusta (80%) and Arabica (20%)
• Robusta is cultivated mainly between 1200-1500 masl.
• While Arabica coffee between 1500-2300 masl
• Uganda is known to produce the best Robusta coffee
• It is the biggest coffee exporter in Africa (low consumer)
• Overall, coffee contributes 20% of Uganda’s foreign
currency earnings
• Coffee is cultivated by mainly small holder farmers, with
average farm sizes of 0.25 hectres.

4. COMPONENTS OF COFFEE RESEARCH IN UGANDA
Leaf
discs/explants
Somatic
Embryos in
test tubes
Mature
embryos in a
bioreactor
ready transfer
Mature
embryos
transferred on
dishes
Plants in
bags ready
for field
Kituza, COREC at 39 Km east of
Kla, on 195Ha
Arabica coffee research, mainly at
Bugusege, in Sironko, 6 Ha
Tissue culture and
germplasm conservation at
Kawanda (NARLI)

5. THE CURRENT COFFEE KILLER CONSTRAINTS
• Coffee wilt disease (CWD)
•Lack of sufficient CWD resistant planting materials
• Black coffee twig borer (BCTB)
•Coffee leaf rust (CLR) – on Arabica coffee
• Water stress (effects of climate change)
• Declining soil fertility
• Weak extension services

6. SOME OF THE BIOTIC KILLER CONSTRAINTS
Coffee wilt disease; Robusta
•Symptoms: Whole plants dry
•Can lose a whole field
Black coffee twig borer
•Twigs killed and dry
•Mistaken to be CWD
•Spreads very fast
Coffee leaf rust
•Plants lose leaves
•Harvested berries
empty shells

7. COFFEE WILT DISEAEASE
RESISTANT ROBUSTA VARIETIES
DEVELOPED
A mother garden of the coffee wilt
disease resistant varieties intercropped
with bananas at COREC. Farmers are
advised to intercrop coffee and bananas
to enhance their income and food
security
One of the coffee wilt disease resistant
variety in the field at COREC

8. MULTIPLICATION OF PLANTING
MATERIALS
1. Seed: Applicable to mainly Arabica coffee
2. Rarely applied with Robusta coffee due to outcrossing
3. Rooted clonal cuttings (Can produce up to 100 plts
per year from one plant)
4. Tissue culture (Can produce up to 10,000 plts per
year from one leaf) or even more than 100,000 per
plant. Have contracted AGT, a private Lab., to
produce up to 2,000,000 plants.

9. EX-PLANTS GENERATED FROM CLEAN LEAVES
Coffee Leaf discs (explants).
Explants on sterile solid medium (right)

10. EMBRYO INDUCTION AND EMBRYO DEVELOPMENT MEDIA
Macro elements
KNO3
NH4NO3
MgSO4 7 H2O
CaCL2 2 H20
KH2PO4 2 H20
mg/l
mg/l
mg/l
mg/l
mg/l
FeS04 7H20
Na2 EDTA 2 H20
Microelements
CuSO4 5 H2O
MnSO4 1 H20
Kl
Na2MoO4 2 H2O
ZnSO4 7 H2O
H3BO3
CoCL2 6 H2O
Vitamins
inositol
nicotinic acid
pyridoxine HCl
thiamine HCl
pantothenic acid
biotine
BAP
Sucrose
Gelrite
Ph
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
mg/l
g/l
g/l
Embryo induction (MS1)
475
412.5
92.5
110
85
13.9
18.65
MS
0.0125
8.45
0.415
0.125
5.3
3.1
0.0125
Gamborg
100
1
1
10
_
_
1.5
30
3
5.7
Embryo development (MS2)
1900
1650
370
440
170
27.8
37.3
MS
0.025
16.9
0.83
0.25
10.6
6.2
0.025
Morel(1)
100
1
1
10
1
0.01
0.5
30
_
5.7

11. DIRECT SOMATIC EMBRYOGENESIS
Somatic embryos growing on media
from ex-plants in test-tubes or Petridishes
Somatic embryos are removed and transferred into bioreactors (RITA) containing liquid
medium for further growth. One ex-plants can yield up to 300 coffee plantlets per year, or
3000 from one leaf. Harvesting of coffee embryos from bioreactors (RITA) can start as early
as 2 months and lasts for a maximum of six months. This implies that sorting of ready
embryos for weaning takes four months.

12. INDIRECT SOMATIC EMBRYOGENESIS
The callus from coffee ex-plants is
cultured in liquid medium in a conical
flask for 3 months to generate embryos
Embryogenic callus is a mass of
undifferentiated cells which are capable
of developing into coffee embryos
The embryos are then transferred to
bioreactors (RITAs) for further growth. With
indirect somatic embryogenesis, callus from
one ex-plant can yield up to 1000 coffee
plantlets per year or 10,000 per leaf.

13. PIERSON MEDIUM FOR INDUCTION OF COFFEE EMBRYOGENIC CALLUS
MACRO NUTRIENTS MS/2
NH4NO3
KNO3
CaCl2 , 2H2O
MgSO4
KH2PO4
Na2 EDTA
FeSO4 , 7H2O
MICRO NUTRIENTS MS/2
H3BO3
MnSO4 , H2O
ZnSO4 , 7H2O
KI
Na2MoO4 , 2H2O
CuSO4 , 5H2O
CoCl2 , 6H2O
VITAMINS Pierson (3)
MYO – INOSITOL
THIAMINE HCl ( B1 )
L-CYSTEINE
CYTOKININE
2 IP
AUXINE
IBA
HYDROLYSAT CASEINE
HYDOLYSAT CASEINE
SUCROSE
SUCROSE
GELING AGENT
Agar
Ph
mg / l
825
950
166
90
85
37.3
27.8
mg / l
3.1
8.45
4.3
0.41
0.12
0.012
0.012
mg / l
100
10
50
mg / l
1
5
mg / l
100
g/l
30
g/l
8
5.7

14. EMBRYOS ARE HARVESTED AND
TRANSFERRED TO THE BIO-REACTORS
Only mature embryos in RITA are sorted for harvesting after they are fully developed.
After they have attained one pair of true leaves, they are removed from RITAs and taken
out side of the laboratory (weaning shade) for conditioning and weaning.

15. CONDITIONING AND WEANING OF
EMBRYOS
Coffee embryos that come out of the laboratory are fragile and very delicate. For this
reason, they need to be conditioned to get rid of excess water to favour the weaning
process. Conditioning is done by spreading the coffee embryos on weaning substrate
(decomposed saw dust or coconut fibres) in transparent plastic boxes for a period of two
weeks. After conditioning, they are planted one by one in weaning medium and kept in a
shade net where they spend an average of 3 month before subjecting them to hardening
conditions.

16. PLANTLETS ON WEANING SUBSTRATES
Weaned young coffee plantlets
Fully germinated coffee plantlets ready
for transplanting into polythene pots

17. PLANTS ARE READY FOR FARMERS
When coffee plantlets have attained 3 to four pairs of true leaves, they are
transplanted into polythene pots containing a mixture of soil and sand.
These are kept in humidity bins for 2 months under 90% shade where they
are maintained by watering, fertilizer application and insect control. The
plantlets are then transferred to 50% shade net where they spend an
additional two months before they are taken by nursery operators.

18. ACKNOWLEDGEMENT
•
•
•
•
•
•
•
•
The Government of Uganda
European Union
UCDA
CFC
CABI
USAID: APEP, LEAD;IPM/CRSP.
AGT
Uganda farmers