GCP 21 II conference Cassava Overcoming Challenges of

GCP 21 -II conference: Cassava: Overcoming Challenges of Global Climatic Changes 18 th-22 nd 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

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 cropsbanana, maize, rice, sweet potatoes, sorghum

• 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

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

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.

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

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

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

• 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 : Øsomatic embryo induction ability Ø sustainability of the S. E ØSubsequently regeneration of S. E to whole plant

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

• Also, transformation vector for transgene delivery was developed: 3 ds. RNAi constructs EACMV i. 2 constructs- Rep/ AC 1 ii. 1 construct- overlapping region of Tr. AP/AC 2 & REn/ AC 3 (AC 2/3) iii. ACMV constructs –still underway ØVector p. GSA 1285 (p. CAMBIA)

Induction of Somatic Embryos (SE) • Young unopened leaves /leaf lobes (2 wks old)- 20 -Cassava landraces–(Farmer Preferred) • MS supplemented with: v 4 -16 mg/L of 2, 4 -D, or Picloram v 2 -6 % (w/v) sucrose v 0 -1. 5 m. M Cu. SO 4 v. Casein hydrolysate • Incubation 28ºC, 16 hours photoperiod for 4 -8 weeks to produce somatic embryo cotyledons.

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 GA 3. • Incubation- 3 to 6 weeks depending on the cultivar

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

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

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

E E E F Root induction and maturation on MS media

2 wks old plantlets in the screen house

3 Cultivars Vs Sucrose and Cu. SO 4 conc.

m to go Ga zi na M a 1 ky ta 0 Ka ki ni No of S. E / total explants 70 Ka bi ba li nd am en o Ki ro ba Ki ng i Ra la ga Sa Cultivars Vs Copper Sulphate conc (m. M) 80 1. 5 60 50 40 30 20 10 0

Regeneration stages achieved by 20 FPC

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

Transformation Vector for Transgene Delivery • RNAi constructs development Øds. RNA vector p. GSA 1285 (p. CAMBIA) • Construct plan: ihp. RNAi with 200 bp or 450 bp arms-sense and antisense • Two: (i) Target-AC 1 and (ii)AC 2/3 partial genes. EACMV

ihp. RNAi construct plan Big. III Hind. III Xho. I Bam. HI Sac. I EACMV (AC 1/5’or AC 2/3) Ca. MV 35 S (3 x) Sense Spe. I EACMV (AC 1/5’ or AC 2/3) Gus intron Antisense OCS terminator

Development of ihp. RNAi constructs • DNA Source: EACMV & ACMV -symptomatic cassava leaves (Delaporta et al. , 1983+ some few modifications) • Specific primers to confirm the above: • Primers: EAB 555/F & R -EACMV DNA B JSP 001 & JSP 002 -ACMV (AV 1/CP)

• Primers design for genes of interest sense + antisense Ref sequences: Gen. Bank AY 795983. 1 • STD cloning - p. GEMT-easy vector • Insert confirmation: Nucleotide. Sequence – Big. Dye™ Terminator technology, Bec. A-ILRI Hub, Nairobi-Kenya

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

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 p. GSA 1285 • Nucleotide analysis BLAST (blastn) program, NCBI

Nucleotide sequence confirmation Similarity to EACMCV-AY 795983. 1 • Construct 1: 98% • Construct 2: 97% Similarity to EACMV-TZM-AY 795985. 1 • Construct 3: 96%

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

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

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

Thank you all!