1. Elibariki, G; Njahira, M; Hosea, K.M.M; Skilton,R; Ndunguru, J
GCP21-II conference: Cassava: Overcoming Challenges of Global Climatic Changes 18th-22nd June- at Speke Resort and Conference Center, Kampala-Uganda
Somatic embryogenesis and development of RNAi constructs for transformation of TZ farmer preferred cassava landraces for CMD resistance
Elibariki, G; Njahira, M; Hosea, K.M.M; Skilton,R; Ndunguru, J

2. Cassava background
A root tuber crop, Euphorbiaceae family
Can strive within almost all climatic conditions
Harsh environs + high Tms, cassava precedes other African staple food crops-banana, maize, rice, sweet potatoes, sorghum

3. Suitable food crop for fight against hunger and poverty in Africa and sub-Saharan continent
TZ among the 10 producers in Africa, total calories per day from cassava =15%
However, among the major challenges facing cassava production is virus diseases.
CBSD and CMD -major concerns

4. Most of TZ farmer preferred cassava (FPC) are highly yielding but are highly susceptible to CMD
CMD incidences up to 72% , 2004
Survey 2005; TZ, 80% of cassava plants showed severe symptoms of CMD (Ndunguru et al., 2005)
2006; CMD alone-47% yield loss

5. However;
Due to the potentiality of this crop, there is a need for more efforts in fighting the viruses.
Genetic transformation (GT) to enhance resistance against viruses affecting cassava is inevitable.

6. Efforts to combat viral diseases-CMD & CBSD
Conventional breeding
Introgression
Phytosanitation
Good Agricultural practices
Biotechonology tools- G.T, genomics, MAs proteomics e.t.c
Reseachers
Farmers
Researchers

7. USA LAB-ILTAB, DANFORTH CENTRE
EUROPEAN LABs
(e.g ETH)
(IITA,NARS)
AFRICA
Further efforts
Technology transfer +
Capacity building
Collaboration
Local farmers/ commercial??

8. Despite other challenges of using biotechnology tools such as GT;
Successful GT of cassava requires
an efficient and effective transformation vectors for transgene delivery
reliable protocols for transgenic plants recovery.

9. Somatic embryogenesis (SE)- among regeneration protocols for organized embryos of cassava
Others:organogenesis,FEC,suspension cultures
This study assessed some selected TZ farmer preferred cassava (FPC) for :
somatic embryo induction ability
sustainability of the S.E
Subsequently regeneration of S.E to whole plant

10. Somatic embryogenesis regime
2. GLOBULAR STAGE
3. HEART
4. TORPEDO
5. COTYLEDONARY
6. MATURED EMBRYOS
7. CONVERSION TO PLANTS
1. CALLUS
EXPLANT
Somatic embryogenesis regime

11. Also, transformation vector for transgene delivery was developed:
3 dsRNAi constructs EACMV
2 constructs- Rep/ AC1
ii. 1 construct- overlapping region of TrAP/AC2 & REn/ AC3 (AC2/3)
iii. ACMV constructs –still underway
Vector pGSA1285 (pCAMBIA)

12. Induction of Somatic Embryos (SE)
Young unopened leaves /leaf lobes (2wks old)- 20-Cassava landraces–(Farmer Preferred)
MS supplemented with:
4-16 mg/L of 2,4-D, or Picloram
2-6 % (w/v) sucrose
0-1.5 mM CuSO4
Casein hydrolysate
Incubation 28ºC, 16 hours photoperiod for 4-8 weeks to produce somatic embryo cotyledons.

13. Incubation- 3 to 6 weeks depending on the cultivar
Maturation of somatic embryos
Globular stage somatic embryos on maturation media (MS salts) +
2% (w/v) sucrose, 1 mg/L thiamine-HCL, 100 mg/L myo-inositol, 0.01 mg/L, 2,4-D, 0.5 mg/L BAP and 0.5 mg/L GA3.
Incubation- 3 to 6 weeks depending on the cultivar

14. Germination and plant recovery
Mature S.E, distinct root and shoot axes on root induction media
Several weeks incubations (up to 6 wks)
Acclimatization, transfer of plants into soil & maintenance in the screen house

15. Results
Generally 65% FPC were capable of S.E
Induction took btn 4 to 8 wks for landraces tested
Different performance under different media optimizations and light vs darkness

16. B A 2 2 SE 4 3
SAGALATO 1
MNAZI D C
Regeneration Regime Of Cassava Landraces Via S.E

17. Root induction and maturation on MS mediaF
Root induction and maturation on MS media

18. 2wks old plantlets in the screen house

19. 3 Cultivars Vs Sucrose and CuSO4 conc.

21. Regeneration stages achieved by 20 FPC

22. Other observations Explant status-health
Leaf lobe age-constant 2wks but some had already opened
Lab and screen house conditions-esp Tm stability
Genotype

23. Transformation Vector for Transgene Delivery
RNAi constructs development
dsRNA vector pGSA1285 (pCAMBIA)
Construct plan: ihpRNAi with 200bp or 450bp arms-sense and antisense
Two: (i) Target-AC1 and (ii)AC2/3 partial genes-EACMV

24. ihpRNAi construct plan
SacI
BamHI
XhoI
SpeI
Gus intron
OCS terminator
CaMV35S (3x)
BigIII
HindIII
Sense
Antisense
EACMV (AC1/5’or AC2/3)
EACMV (AC1/5’ or AC2/3)

25. Development of ihpRNAi constructs
DNA Source: EACMV & ACMV -symptomatic cassava leaves (Delaporta et al., some few modifications)
Specific primers to confirm the above:
Primers:EAB555/F & R -EACMV DNA B
JSP001 & JSP002-ACMV (AV1/CP)

26. Ref sequences:GenBank
Primers design for genes of interest sense + antisense
Ref sequences:GenBank AY
STD cloning - pGEMT-easy vector
Insert confirmation: NucleotideSequence –BigDye™ Terminator technology, BecA-ILRI Hub, Nairobi-Kenya

27. Cloning into an Expression vector
Appropriate fragments released from pGEMT-easy vector were cloned –into an expression vector
Screening of putative E. coli transformants
Colony PCR (bacterial)
Plasmid isolation/purification
Restriction digestion
Nucleotide sequence analysis

28. + Nucleotide analysis……
Pair of primers used for synthesis of respective fragments in PCR was used in sequencing +
Primers designed –flanking upstream and downstream of inserts/MCSs of the vector pGSA1285
Nucleotide analysis BLAST (blastn) program, NCBI

29. Nucleotide sequence confirmation
Similarity to EACMCV-AY
Construct 1: 98%
Construct 2: 97%
Similarity to EACMV-TZM-AY
Construct 3: 96%

30. Conclusion SE regeneration platform system now in place at MARI
Plantlets acclimatization- difficulty, still need some optimizations
Frequency of SE cotyledons and % recovery of SEs was good
Variables -might be genotype specific…(genotype analysis ongoing)

31. Perspectives
Constructs suitability & efficiency evaluation through GT of some few selected cultivars (FPC)
GT- Agrobacterium mediated via S.E
Further analysis
Recommendation
More capacity building needed for R&D in LDCs –especially young Scientists & more collaboration

32. Acknowledgments
UDSM staff Dev Fund & UDSM CoNAS World Bank Project- PhD Sponsor
MBB Dept-UDSM
TZ govt ,Gates, Rockfeller Found, ILTAB, ASARECA +ALL donors-MARI Biosafety lab (II)
MARI – Admin & Staff
BecA –ILRI Hub & ABCF
Supervisors: KMM Hosea, R. Skilton & J.Ndunguru
The Ohio State Univ for expression vector to Dr Joseph
USAID

33. Thank you all!