Empowering biotechnology in southern Africa: Establishment of a robust transformation platform for the production of industry preferred cassava Chrissie Rey 1, Chezlyn Chetty 1, Claudia Rossin 1, Sarah Rogans 1, M Moralo 1, W Gruissem 2 and Herve Vanderschuren 2 1 Plant Biotechnology Programme School of Molecular and Cell Biology University of the Witwatersrand 2 Institute of Plant Sciences, ETH Zurich, C C Chetty, C B Rossin, W Gruissem, H Vanderschuren and M E C Rey New Biotechnology (published online 7 June 2012)

Hang out with an impala!

And to go with your beer

CASSAVA IN SOUTHERN AFRICA: FOOD, FEED AND STARCH Tonga tribesman growing cassava for hundreds of years Grown in SA, Swaziland and Mozambique for food security with maize; Casquip Starch Man.: commercial farm Potential of cassava for industrial starch (food, textile and paper) and bioethanol SAPPI and MONDE: paper SA Breweries for beer (Impala) T200: industry preferred high-starch landrace Cassava mosaic disease (CMD): most significant constraint of cassava production in southern Africa

CASSAVA IN SOUTHERN AFRICA: FOOD, FEED AND STARCH Tonga tribesman growing cassava for hundreds of years Grown in SA, Swaziland and Mozambique for food security with maize Potential of cassava for industrial starch (food, textile and paper) and bioethanol SAPPI and MONDE: paper SA Breweries for beer (Impala) T200: industry preferred high- starch landrace Cassava mosaic disease (CMD): most significant constraint of cassava production in southern Africa

Challenges for GM virus-resistant cassava Are 7 distinct viral species with limited sequence homology; three of these (ACMV, EACMV and SACMV) occur in SA High virus recombination rates lead to fast generation of biodiversity Mixtures of viruses in the field Uncontrolled movement of infected stakes between geographical regions (vegetative virus spread) High genetic diversity of the whitefly vector: SSAF 1-5; SSAF 1 main WF on cassava

Challenges for cassava transformation Need to engineer cassava for virus resistance and other traits Cassava is highly recalcitrant to transformation; transformation efficiencies low at ±5 - 15% Transformation system lacks reproducibility & flexibility; labor- intensive and fastidious Regeneration potential is also low Robust and reproducible system only for model lab cultivar 60444; limited success for farmer-preferred landraces or cultivars Few laboratories in the northern hemisphere have achieved success; no sustainable & reproducible platform/pipeline on African continent Knowledge & technology transfer to African labs offering less optimal conditions is needed

IMMUNITY: SCREEN FOR R GENES RECOVERY: siRNA CASSAVA RESEARCH PROGRAM TRANSGENICS: PTGS (siRNA & amiRNA) RESISTANCE BREAKING SATELLITES VIRUS DIVERSITY AND EVOLUTION WHITEFLY BIOTYPING AND GENOMICS CISGENICS SACMV EACMV ACMV

South African cassava mosaic virus Gene targets for silencing AC1- replication associated protein AC4 – silencing of host PTGS/symptom determinant AC2/AC3 - replication enhancer/ transcription enhancer BC1- cell-to-cell movement protein  Chimeric tandem constructs of ACMV, SACMV and EACMV

Approaches to genetic engineering  Antisense RNA  Hairpins (inverted repeats) to induce antiviral siRNAs (hpRNAi)  Inverted repeats with mismatches in the sense strand  Artificial miRNAs  Defective interfering molecules

Intron-containing long IR constructs not always stable: DNA in IR conformation can form 4-way helical junctions (cruciforms) Cruciforms are unstable & are key intermediates for homologous recombination Introduced bp mismatches (C - T) in the sense arm of SACMV Rep (AC1) & BC1 IRs, by sodium bisulphite treatment, preventing cruciforms, thermodynamic stabilization of DNA secondary structure and resulting RNA hairpin Filed PCT in Europe; SA patent Biotechniques 52(4):

Mismatched SACMV AC1 Real time qPCR results in Nicotiana benthamiana shows viral knockdown & reduced symptoms 3 classes of siRNA detected: 21, 24, and nt

miRNAs siRNAs RNA SILENCING Virus derived transgene silencing constructs Antisense Hairpins that produce siRNA (NMM;MM) amiRNA processed to produce antiviral siRNAs Agrobacterium-mediated or particle bombardment transformation of TMS60444 & T200 Somatic Embryo (SE) Friable Embryogenic callus (FEC) Developing somatic embryo Cotyledo n Transgenic plantlet GUS FEC GUS cotyledon Molecular Analysis MODIFIED METHOD DEVELOPED BY ETH ZURICH 8 months & 10 months for T200 ILL AB

Somatic Embryogenesis (SE) 3 cycles (6 weeks)10 weeks (5 cycles) SE cycles to generate friable embryogenic (FEC) 6 weeks (light)8-10 weeks (dark) FEC proliferationHighMedium/high Quality of FECsExcellentGood No. Transfers to CEM +C100 medium for shooting induction 24 cv T 200 Comparison of transformation process between model cultivar and T200, a SA landrace

Regeneration of cotyledons from wild-type untransformed FECs

Data for transformation, and regeneration of transformed FECs, for TMS and T200 Cumulative results from two parallel experiments, each starting with 70 FEC clusters. a Total number of putatively transformed cotyledons emerging from 140 FEC clusters. b Calculated as the number of shoots obtained on CEM divided by the number of putatively transformed cotyledons. c Calculated as the number of shoots that produced roots on selection media divided by the number of shoots that developed on CEM (represents percentage of transformed rooted/ shooted plantlets derived per plate per five FEC clusters). d. Number of transgenics from 140 FEC clusters. CultivarNo. germinated cotyledons a Shooting efficiency (%) b Percentage rooted plantlets c Transformation efficiency d cv T

Summary High transformation efficiencies for cv (45%) and SA cultivar T200 (33%); shooting of T200 needs improvement We have 6 acclimatized transgenic lines in greenhouse trials (SACMV BC1; AC1/AC4 SACMV (MM and NM) and AC1/AC4 & AC2/3 ACMV in TMS & T200) Testing other siRNA and amiRNA stacked constructs Constructed and testing minimal cassettes by biolistics for commercialization This research was a collaboration between Wits and ETH Zurich and emphasizes the importance of technology transfer between the hemispheres There is a need to establish CAPACITY and a sustainable, robust and reproducible CASSAVA TRANSFORMATION PLATFORM for GE on the African continent for other traits such as drought, nutritional fortification

Challenges Capacity training of a sustainable technical expertise base of critical mass Cassava transformation genotype dependent; 8-10 months to generate FEC Funding to set up a sustainable and robust cassava transformation pipeline/platform Despite GM legislation, process is slow, especially for new crops Field trial permit; biosafety and risk assessments Lack of expertise in SA and continent for commercialization Intellectual Property (IP) still faces challenges Private sector investment: commercial farmers Also target small-scale farmers

UNIVERSITY OF THE WITWATERSRAND Thank NRF, Casquip Starch Manufacturing Co. and TIA for funding; and for support from the SA-Swiss S&T Bilateral Programme

RNA SILENCING: PLANT INNATE IMMUNITY