Tissue Culture,Crop Genetic Transformation,Molecular marker applications,Capacity Building & partnerships

1. Biotechnology
Research-for-Development
at IITA
I Ingelbrecht
IITA-Ibadan, 31 August 09

2. Guiding principles
 Biotech complementary tool to conventional approaches
 Multidisciplinary approach (breeding, genetic resources,
pathology, etc)
 Product oriented
 Partnerships: national research programs across SSA
and advanced labs overseas
 Integrate capacity building with research

3.  ~8 full time PhD scientists with additional scientists
(breeding, virology, pathology, etc)
 Two main hubs: HQ in Nigeria + BECA, Kenya

4. Biotechnology R-4-D
‘traditional’ ‘modern’
In vitro Tissue Culture Crop Genetic Molecular Marker
Transformation Applications
Capacity building

5. I. In vitro Tissue Culture
yam
Banana, plantain
cassava

6. Activities
 In vitro micropropagation for distribution of elite
cassava, yam and banana/plantain materials
 Disease clean up: production of certified, disease-free
planting materials (with PQS)
 Training + technical backstopping of NARS in all
aspects of in vitro micropropagation and set up of
tissue culture facilities

7. Impact
 High demand by national programs across SSA
 Technology well established: applications for breeding &
genetic resource conservation (fairly) routine
 Technology transferred to national programs, in some cases
(high value crops) applied for commercial purposes
In vitro tissue culture recently integrated with Genetic
Resources Center at HQ, Ibadan, Nigeria

8. II. Crop Genetic Transformation
• GM crops developed and commercialized in industrialized as well as
developing countries with demonstrated benefits for producers and
environment
• With proper identification of targets, GM technology can complement
conventional breeding for intractable traits in SSA agriculture
• For ‘orphan’ crops tools and products mostly in stage of development,
some already available
• Need to consider regulatory environment lacking in most countries
in SSA; capacity building

9. Ongoing projects
Genetic transformation of cassava for resistance to the
Cassava Brown Streak Disease, starch modification
Genetic transformation of banana for bacterial blight
resistance,
DNA recombinant technology for construction of genes
and analysis of transgenic plants
Biosafety with application for CFT of GM crops, by
national programs

10. Example: cassava, banana
Three steps in genetic transformation protocol:
1. In vitro shoot regeneration method
2. Gene transfer method: Agrobacterium
3. Selection and/or screening for transgenic shoots
Focus on African genotypes

11. Transformation of farmer-preferred cassava
for CBSD resistance
Equatorial
Guinea Uganda
Kenya
ROC
DRC
Tanzania
Malawi
Zambia
Mozambique
CBSD devastating
CBSD damaging
CBSD reported
A transgenics approach for resistance to Potyviruses previously used and
grown commercially: eg papaya resistant to Papaya ringspot virus

12. Why target cassava landraces?
Current transformation protocols are for ‘model’ genotypes, not used
by farmers or breeders in Africa: excellent research tool but
limited application in the field
Bottleneck since current protocols are genotype-dependent
develop protocol for (African) farmer-preferred lines

13. Summary cassava regeneration responses
Explants; e.g. immature leaf lobes
Embryogenesis +/-
Shoot organogenesis
Secondary SE *
Primary
SE
++++ ++++
Multiple shoots
++++
Embryogenesis
++ Cotyledonary Friable embryogenic
stage SE * Callus *
Adventitious shoots
Embryos Protoplasts
+/- Embryogenic
+/- 0 - 40% suspensions
Plantlets ++ 20 - 40%
Fig. modified from Zhang et al. 2006 ++++ >90%

14. Somatic embryogenesis
SE produced for 9 genotypes: 4 landraces from ESA
4 landraces from WCA
1 IITA elite line
cv Albert TME12

15. Transgenic African cassava with reporter gene

16. Transgenic African cassava with reporter gene
a
petiole
4 mm
b
Young stem
3 mm
c
Old stem
5 mm
d
Fibrous root
3 mm
e
tuber
1 cm
Non transgenic Transgenic – 1st ratoon Transgenic – 2nd ratoon

17. Status
- Resistance genes developed and tested in N benthamiana:
resistance is strain specific
- Transformation of African cassava with resistance genes
ongoing

18. Banana transformation - status
- Transgenic plants produced with resistance gene(s) for
bacterial blight
- Field trial planned for 2010 (Uganda)

19. III. Molecular marker applications
Rationale
• For characterization and management of germplasm and pathogens
(diversity) and for diagnostics purposes
• In marker assisted selection/breeder to accelerate breeding
process; extensively used in cereals and other crops of developed world
• As with GM technology, for ‘orphan’ crops tools are mostly in stage
of development

20. Ongoing projects
 Characterization of germplasm collections of cassava, yam,
banana
 QTL mapping of virus (CMD & CBSD) resistance in cassava,
drought tolerance in cowpea, maize and cassava
 MAS for striga resistance in cowpea
 Development of SSR and SNP markers for cassava & yam
 Diagnostic markers for bacterial blight + other pathogens
 Development of DNA microarray for gene discovery in
cassava

21. Example: EST-SSR marker development
Total number of EST sequences investigated: 18,166
Number of unigenes used for in silico identification of SSRs: 8,577
Total number of unique SSR loci appropriate for primer modeling: 646 (3.3%)
Number of candidate SSR investigated : 346
PCR successful: ~ 90% Failed PCR: ~ 10%
PCR products with expected sizes Amplification of introns
> 500 bp
Eliminate
Screen on diversity panel

22. Example cassava
- 3078 cassava accessions with minimal passport data held in trust in GRC
DNA fingerprinting for effective
management and use by breeding
program

23. New Cassava SSR markers
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 23 24
180 new SSR markers
Used for fingerprinting collection, mapping and variety identification

24. Semi-automated genotyping using fluorescent labeling
Different technologies can be used
for diversity studies, QTL mapping

25. Example: cassava microarray
~14,000 genes or 1/4 to
1/3 of all cassava genes

26. - Hybridization and scanning
- Identify differentially expressed genes for use in transgenic
program or complement QTL mapping

27. IV. Capacity Building & partnerships
- Degree (MSc, PhD) and non-degree training of students or VS;
mostly integrated with ongoing research activities; topical also possible if funded
- Workshops: genetic transformation, molecular breeding, biosafety
- Infrastructure: eg MARI, Tanzania for GM
Advanced labs:
DDPSC, USA; ETH, Switzerland; KU, Copenhagen, Univ Virginia, USA; others
National research institutions and universities
NRCRI, Nigeria; MARI, Tanzania, NABDA, Nigeria; many universities in Nigeria
and other African countries for joint degree research.

28. Development investors
 USAID
 RF
 BMGF
 Generation CP
 AATF
 Gatsby Charitable Foundation
 Masterfoods