Presentation at Biosciences For Farming in Africa, Kampala 1st Nov., 2012

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, UGANDAP.O.Box 185, Mukono-KituzaEmail:afrikangire@gmail.com

COFFEE RESEARCH CENTRE, AT KITUZA, MUKONO.

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.

Arabica coffee research, mainly at

Bugusege, in Sironko, 6 HaKituza, COREC at 39 Km east of

Kla, on 195Ha

Tissue culture and

germplasm conservation at

Kawanda (NARLI)

COMPONENTS OF COFFEE RESEARCH IN UGANDA

Leaf

discs/explants

Somatic

Embryos in

test tubes

Mature

embryos in a

bioreactor

ready transfer

Plants in

bags ready

for field

Mature

embryos

transferred on

dishes

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

SOME OF THE BIOTIC KILLER CONSTRAINTS

Coffee wilt disease; Robusta

•Symptoms: Whole plants dry

•Can lose a whole field

Coffee leaf rust

•Plants lose leaves

•Harvested berries

empty shells

Black coffee twig borer

•Twigs killed and dry

•Mistaken to be CWD

•Spreads very fast

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

COFFEE WILT DISEAEASE RESISTANT ROBUSTA VARIETIES DEVELOPED

One of the coffee wilt disease resistant

variety in the field at COREC

MULTIPLICATION OF PLANTING MATERIALS

1. Seed: Applicable to mainly Arabica coffee

2. Rarely applied with Robusta coffee due to out-

crossing

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.

EX-PLANTS GENERATED FROM CLEAN LEAVES

Coffee Leaf discs (explants). Explants on sterile solid medium (right)

Macro elementsEmbryo induction (MS1) Embryo development (MS2)

KNO3 mg/l 475 1900NH4NO3 mg/l 412.5 1650MgSO4 7 H2O mg/l 92.5 370CaCL2 2 H20 mg/l 110 440KH2PO4 2 H20 mg/l 85 170

FeS04 7H20 mg/l 13.9 27.8Na2 EDTA 2 H20 mg/l 18.65 37.3Microelements MS MSCuSO4 5 H2O mg/l 0.0125 0.025MnSO4 1 H20 mg/l 8.45 16.9Kl mg/l 0.415 0.83Na2MoO4 2 H2O mg/l 0.125 0.25ZnSO4 7 H2O mg/l 5.3 10.6H3BO3 mg/l 3.1 6.2CoCL2 6 H2O mg/l 0.0125 0.025Vitamins Gamborg Morel(1)inositol mg/l 100 100nicotinic acid mg/l 1 1pyridoxine HCl mg/l 1 1thiamine HCl mg/l 10 10pantothenic acid mg/l _ 1biotine mg/l _ 0.01BAP mg/l 1.5 0.5Sucrose g/l 30 30Gelrite g/l 3 _Ph 5.7 5.7

EMBRYO INDUCTION AND EMBRYO DEVELOPMENT MEDIA

DIRECT SOMATIC EMBRYOGENESIS

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.

Somatic embryos growing on media

from ex-plants in test-tubes or Petri-

dishes

INDIRECT SOMATIC EMBRYOGENESIS

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.

Embryogenic callus is a mass of

undifferentiated cells which are capable

of developing into coffee embryos

The callus from coffee ex-plants is

cultured in liquid medium in a conical

flask for 3 months to generate embryos

PIERSON MEDIUM FOR INDUCTION OF COFFEE EMBRYOGENIC CALLUSMACRO NUTRIENTS MS/2 mg / lNH4NO3 825

KNO3 950

CaCl2 , 2H2O 166

MgSO4 90

KH2PO4 85

Na2 EDTA 37.3

FeSO4 , 7H2O 27.8

MICRO NUTRIENTS MS/2 mg / lH3BO3 3.1

MnSO4 , H2O 8.45

ZnSO4 , 7H2O 4.3

KI 0.41

Na2MoO4 , 2H2O 0.12

CuSO4 , 5H2O 0.012

CoCl2 , 6H2O 0.012

VITAMINS Pierson (3) mg / lMYO – INOSITOL 100

THIAMINE HCl ( B1 ) 10

L-CYSTEINE 50

CYTOKININE mg / l2 IP 1

AUXINEIBA 5

HYDROLYSAT CASEINE mg / lHYDOLYSAT CASEINE 100

SUCROSE g / lSUCROSE 30

GELING AGENT g / lAgar 8

Ph 5.7

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.

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.

Weaned young coffee plantlets Fully germinated coffee plantlets ready

for transplanting into polythene pots

PLANTLETS ON WEANING SUBSTRATES

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.

PLANTS ARE READY FOR FARMERS

ACKNOWLEDGEMENT

• The Government of Uganda

• European Union

• UCDA

• CFC

• CABI

• USAID: APEP, LEAD;IPM/CRSP.

• AGT

• Uganda farmers