G. Arinaitwe, H. Khana, P. Namanya, . Kiggundu, A W.K Tushemereirwe and J. Dale

Research Framework

National Research Laboratories - Kawanda

Genetic modification of highland bananas: biofortification with PVA and iron

Introduction

�Uganda has a population of 34 M people & is projected to be 53 M by 2025

Problems:

�Inadequate food quantities

�Imbalanced nutrients,

containing mainly

carbohydrates

Banana is synonymous to food

A B C

Bananas in Uganda

Banana weevils

Nematode effect

Banana bacterial wilt

Black Sigatoka disease

Micronutrient deficiencies in Uganda

Micronutrient undernutrition

� The major micronutrient deficiencies in Uganda include:�Vitamin A deficiency (VAD) �Iron deficiency Anaemia (IDA) �Iodine Deficiency Disorders (IDD)

� National Nutritional Surveys 1994-1999�31 out of 36 districts had VAD�50% of children have VAD�40% of children > 5 years have IDA�50% of pregnant women had IDA�30% of maternal deaths were attributable to IDA

psyB73

crt I

psy2 Psy2a

psy

Sources of genes for biofortification

Constructs used in biofortification

1st Batch 2nd Batch(Opt series)

3rd Batch(GEN series)

E1-ZmPsy1 E1-APSy2a E1-ZmPsy1Q60

E1-Crt1 Acs-ZmPsy1Q60+E1-S.Fer E1-APsy2a

E1-APsy2a Acs-ZmPsy1Q60 BT4act-APsy2a

E1-S.Fer E1-ZmPsy1Q60 Ubi-APsy2a

35S-Fro2 E1-ZmPsy1 Ubi-ced9

35S-IrtI Ubi-mced9 Ubi-AtBag4

Ubi-Gus

E1-Gus

Biofortified product for low and highlands

Cultivars: ‘Nakinyika’ (EA-AAA)And ‘Sukali Ndizi’ (ABB)

Cultivars: Hybrid M09Tolerant to: Black Sigatoka, Nematodes and weevils

ECSs work at Kawanda, Uganda

8. Weaning and potting of plantlets

2. Inoculation stage

3. Embryogenic callus

7. Multiple shoot formation and rooting

4. Cell in suspension

5. Somatic embryos

formed from suspensions

1. Male bud

6. Embryo germination

Genetic transformation

Materials and methods

ECS initiation and transformation

ECSs

Selection

Embryogenic callus

ECS infection

Shoots from EC clones

PCR analysesMorphological analyses Southern blot analyses

Male bud Immature flower

Future activities

NARO-KARI Biotechnology Laboratory

Shoot regeneration improved

0.5 mg/L GA3 1.0 mg/L GA3 1.5mg/L GA3Control: 0mg GA3

2 Weeks 1.5 Months 2.5 MonthsTra

nsf

orm

atio

nan

d s

elec

tio

n

Shoot regeneration

Weaning and hardening

First CFT at NARO, Uganda

CFTs at NARO-Kawanda

� Several CFTs of transgenic lines containing disease resistance genes and micronutrient traits

Phenotypes observed

Cultivar %Water contentBCE (µg/g DW)

Day1 Day7 Day 14

Mbwazirume 76.3 9.8 17.5 23.5

Nakinyika 73.9 13.5 21.0 26.5

Nakitembe 76.9 19.6 23.4 28.3

Kisansa 75.1 9.1 18.0 18.8

M9 74.5 4.3 11.0 16.8

Butobe 58.2 18.2 22.3 23.1

Kayinja 65.2 0.8 1.7 2.2

Carotenoid content at FG during storage

NAKINYIKA HYBRID M9 KAYINJANAKITEMBE

High carotenoid content in transgenic S. NdiziHigh carotenoid content in transgenic S. Ndizi

1

2

3

4

Control line ZmPsy1 (12-39) lineCross section of the control and the ZmPsy1 (12-39) line; above: Chromatogram peaks for 12-39 fruit extract identified at λmax=450nm, peak 1, lutein at 6min; peak 2, α-carotene at 12.2; peak 3, trans-β-carotene 13.1min & peak4, cis β-carotene at 13.9min

PVA retention in target cooking bananas

Steamed

Boiled

FOC symptoms of the diseaseYellow leaves

Discoloured pseudostem

Discoloured corm

� Microconidia entry to the corm is through wounded /cut root hairs

� The conidia spreads up the xylem and blocks water and nutrient movement

� Death of plant releases the dormant chlamydospores back into the soil

� Reinfection is through running water and propagating suckers

ND control ND 1 ND 2 ND 3 ND 4

ND control ND 1 ND 2 ND 3 ND 4

FOC wilt in cultivar Sukali Ndizi

1212 55 72 83 NTC

Acknowledgements

NARO

A. Kiggundu G. Arinaitwe

P. LamwakaB. Magambo

R. Ecodu

P.Namanya

QUT

Rob HardingDoug Becker

Marion BatesonJason Geijskes

Harjeet KhannaAnthony James

Jen Kleidon

Bulu Malazi

Prof James Dale (Program Leader)

Dr. WilberforceTushemereirwe