Taming the. Sweetpotato Weevil through Biotechnology. Improving African food staples:

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1 Improving African food staples: Taming the The Sweetpotato Action for Security and Health in Africa (SASHA) is a five-year initiative designed to improve the food security and livelihoods of poor families in Sub- Saharan Africa by exploiting the untapped potential of sweetpotato. It will develop the essential capacities, products, and methods to reposition sweetpotato in food economies of Sub-Saharan African countries to alleviate poverty and under-nutrition. Sweetpotato Weevil through Biotechnology SWEETPOTATO ACTION FOR SECURITY AND HEALTH IN AFRICA

2 Background and justifications Survey on the socio-economic impact of weevils in Uganda (2007-8) reports an average yield loss of over 28% between wet and dry seasons (Kiiza et al., 2009). IPM practices are difficult to implement in small-scale field with sweetpotato grown year round; Effective weevil resistance has not been found and used by conventional breeding; Bt crops have shown to be safe and have a positive impact on the environment while controlling targeted pests due to pesticide use reduction. Kiiza et al., Uganda Country Report

3 Coleopteran pest resistant crops Colorado potato beetle resistant potatoes produced by inserting the cry3a gene from Bacillus thuringiensis (subsp. Tenebrionis) released in by Monsanto. Corn rootworm-resistant maize produced by inserting the cry34ab1 and cry35ab1 genes from Bacillus thuringiensis strain PS149B1 released in 2005 by Dow. Corn rootworm resistant maize produced by transformation with a modified cry3a gene released in 2007 by Syngenta. Corn root worm resistant maize produced by inserting the cry3bb1 gene from Bacillus thuringiensis subsp. Kumamotoensis released in 2001 by Monsanto.

4 What is the final product? Weevil resistant sweetpotato: Resistance against African weevils Cylas puncticollis and C. brunneus (and C. formicarius, Blosyrus obliguatus) Stability: from root bulking until harvesting under broad environmental conditions Efficacy: larvae mortality of >99% within the first 5 days after hatching under field conditions (no tunneling) Durability: two WR genes (DG) expressing proteins active against weevils with non-competitive binding properties Adoption: variety with good acceptability adapted for region where weevil damage is severe Availability: no contractual, IPR, or regulatory restrictions for use in SSA countries

5 Outline Objective 1: Identification of most active proteins against African weevils [NaCRRI, UG; Auburn University, USA; CIP, PE] Objective 2: Gene constructs with freedom to operate in SSA countries [CIP, PE] Objective 3: Weevil Resistant Variety development: Mode of action of the Cry proteins [University of Valencia, SP] Direct transfer into African variety [CIP, PE; CIP/BecA, KE; Kenyatta University, KE; NaCRRI, UG] Transgenic breeding [CIP, PE; UPRM, USA; NaCRRI, UG] Objective 4: Safety to human and the environment [D Danfort PSC, USA] Objective 5: Capacity building, knowledge sharing communication [Ghent Univ., BE]

6 Cry proteins active against weevils LC 50 values (mg/ml diet) of Bt proteins against 1st instar C. puncticollis and 2nd instar C. brunneus (An LC 50 below 1 ppm is low enough to expect high levels of toxicity when expressed in sweetpotato) Cylas puncticollis Cylas brunneus Bt Total LC50 95% Slope Total LC50 95% Slope protein n Values F.L (mean ± SE) χ 2 n Values F.L (mean ± SE) χ 2 Bt1 (ET33/34) ± ± Bt2 (ET70) ± ± Bt3 (Cry3Aa3) ± ± Bt4 (Cry3Bb2) ± ± Bt5 (Cry3Bb3) ± ± Bt6 (Cry3Ca1) ± ± Bt7 (Cry7A1) ± ± Moses et al. (2010). Journal of Economic Entomology 103:

7 Mode of Action of the Cry proteins University of Valencia - Spain: B. Escriche, J. Ferre, M. Martinez Cry proteins are Cry7Aa1, Cry3Ca1, and ET33-34 Bacillus thuringiensis strains obtained for Cry7Aa1 and Cry3Ca1: Cry7Aa1 protein produced from this strain was processed into a larger fragment and did not bind to receptor of the weevils more protein / strain characterization underway Cry3Ca1 protein produced from this strain was processed into a fragment of the expected size and bound to receptors of weevils. However, this binding appears non-competitive. Fig.: Crystal proteins produced by Bacillus thuringiensis with activity against insect pests

8 Gene construct design No contractual /IPR restrictions for use in SSA countries: Public domain genic elements; re isolation and use in countries where patents are not registered; or chemical synthesis Sweetpotato like genes: Cry proteins are chosen based on efficacy and lack of homology with known allergens and toxins Fragment with activity optimized for codon usage in sweetpotato Promoter and polyadenylation sequences of SP genes with high expression in roots and induced by wounding: sporamin and b amylase genes Single gene constructs: Identify the best WR gene(s) of the three candidates (efficacy / homology). Primarily for proof of concept. Double gene constructs: Will allow us to transfer two genes at the same time (during transformation) as part of the insect resistance management strategy. Primarily for the final product.

9 Intellectual property plan Search of relevant patents encompassing the use of 3 Cry proteins Cry7Aa1, Cry3Ca1, and CryET33/CryET34 in sweetpotato in Peru, Kenya, Uganda, and member states of OAPI and ARIPO was done by IP students oriented by two professors at the Pierce Law Center (PIPRA): None appeared to be registered in Peru and SSA countries At the request of the B&M GATES Foundation, PIPRA made a review and highlighted 3 issues needing clarity: Two new MTAs were obtained One patent lawyer opinion obtained Conclusions: the WRSP technology has freedom to operate in SSA countries

10 5 WR gene constructs 3 SG and 2 DG (cry7aa1 assumed to be the best one) WR gene 1 WR gene 2 Selectable marker Backbone vector pcip -Amy:cry7Aa1 none nptii pcambia pcip78 gspoa1:cry3ca1 none nptii pcambia pcip79 gspoa1:et33-34 none nptii pcambia pcip82 -Amy:cry7Aa1 gspoa1:cry3ca1 nptii pcambia pcip84 gspoa1:et Amy:cry7Aa1 nptii pcambia pcip85 nptii T35S POLY A SITE T BORDER (L) kanamycin (R) nos P gspoa1 promoter Cry3Ca1 nos P nptii T35S POLY A SITE T BORDER (L) kanamycin (R) pbr322 ori gspoa1 promoter Cry3Ca1 3' gspoa1 pbr322 ori pbr322 bom site pcip bp 3' gspoa1 T-BORDER (R) pbr322 bom site pvs1-rep pcip bp B-amyl TATA box Cry7Aa1 pvs1-rep pvs1 Sta pvs1 Sta 3' beta-amy T-BORDER (R)

11 Direct transfer into African varieties Two strategies for sweetpotato transformation: Target tissue Gene transfer Organogenesis Selection and regeneration In vitro mother plants of cv Jewel top 3 leaves with petioles callus induction 3-5 days shoot formation A. tumefaciens mediated transformation 24 h 3-5 weeks Transgenic plantlet Embryogenic In vitro mother plants of cv Huachano top 3 leaves with petioles Callus Induction Embryo development A. tumefaciens mediated transformation 24 h months callus formation months 1-3 month Plant development 1-2 months Transgenic plantlet In vitro mother plants of cv Jonathan top 3 lateral meristems Callus induction Embryogenic 2 months (dark) Embryo development 1-3 month Plant development 1-2 months Transgenic plantlet A. tumefaciens mediated transformation 24 h callus formation months Somatic embryo genesis

12 Direct transfer into African varieties Organogenesis (ABL and BecA): Out of 31 African varieties, 6 had regeneration efficiencies higher than 40% [Mugande, Imby, Luapula, Kawogo, Zambezi and Mafutha] Ukerewe and Luapula have so far generated 2 putative transformed events using pcip85. Conclusions: 1. From this study, we would conclude that approximately 20% of African sweetpotato cultivars are amenable to transformation by organogenesis; 2. However, the yield is still too low to be consider as the transformation method of choice for commercial product development.

13 Direct transfer into African varieties Somatic embryogenesis (ABL and Kawanda): At ABL, the variety Imby from Burundi produced 31 independent events through s.e. after 8 to 12 months; At Kawada, out of 11 Ugandan cvs established in vitro and regenerated through s. e., 6 produced embryogenic callus, and 4 of them roots from callus Conclusions: Large number of independent events can be produced but better African varieties need to be identified.

14 Transgenic Breeding Rationale: Genetic transformation has been mostly successful with non-african varieties [Jewel, Huachano, Jonathan] The identification of one event with high accumulation of Cry proteins causing high mortality of weevils offers the option for crossing with African germplasm (=Transgenic breeding). This event should ideally be accumulating 2 Cry proteins with independent modes of action, single copy TDNA, no other foreign DNA, and not interrupting or creating new ORF. End-result is an approved event in a diverse germplasm ready for selection under different agro-ecologies and for different uses.

15 Transgenic Breeding In total at ABL, 90 transformed events have been obtained by organogenesis and somatic embryogenesis: Gene Binary plasmid Jewel Wagabolige Huachano Imby Mugande* Jonhatan cry7aa1 pcip nd nd nd nd cry3ca1 pcip79 9 nd nd nd nd nd ET33-34 pcip82 8 nd nd nd nd nd cry7aa1 + cry3ca1 pcip84 6 nd cry7aa1 + ET33-34 pcip85 2 nd 5 nd nd nd The events from Jewel (30) have been characterized in details

16 Transgenic Breeding Relative expression plot of WRSP genes in leaves of transformed events from Jewel: Identification of high expressers for each cry gene. Fig.: Each assay was conducted using real time PCR and primers specific to each transgene. The Y axis represents the relative amount with respect to the lowest expresser event. The X axis represents the events

17 Transgenic Breeding Relative accumulation plot of Cry proteins in leaves of transformed events from Jewel: Quantification of Cry protein accumulation in leaves, skin and flesh of the storage roots; No correlation between transcription in leaves and Cry protein accumulation in the storage roots; Level of accumulation in the storage root flesh are for all but two inferior to LC50 (<1ppm). Fig.: Each assay was conducted using antibodies against the pro-toxin and are represented here as mg per gr fresh weight (Y axis). The X axis represents the events

18 Transgenic Breeding Best candidate events for resistance assays are: CIP cry7aa1 CIP cry3ca1 CIP ET33-34 CIP cry7aa1 + cry3ca1 CIP cry7aa1 + ET33-34 Storage roots produced at BecA and KU from 29 events from Jewel

19 Efficacy against African weevils Assay 1 Artificial diet infested by 2 nd instar larvae: delicate handling of larvae, high mortality, 8% DM, results with no statistical significance;

20 Efficacy against African weevils Assay 2 Whole root infested with female adults: need large number of samples and contrasting resistance levels due to high variability among female laying eggs; no significant differences.

21 Efficacy against African weevils Assay 3 Root chips infested by plug with one egg: difficult to control infection; Abandon as difficult to determine mortality causes.

22 Efficacy against African weevils Assay 4 Small roots infested by plug with one egg: Improvement over the root chip method; Plug of non transgenic flesh with two 24 hr eggs infested onto the transgenic root; After 18 days, CIP had larvae mortality in 2 reps (event with highest Cry [ET33-34] protein accumulation in the storage root); Need additional repetitions.

23 Transgenic breeding crossing Crossing: Events with high accumulation of Cry protein (CIP , CIP , CIP , CIP and CIP ) grafted to I. Setosa to induce flowering; Crossing underway with New Kawogo, Tanzania and Naspot 1;

24 Safety to human History of safe use // Familiarity Protein allergenicity: assayed using two web search tools: SDAP - Structural Database of Allergenic Proteins and Allermatch. For both cases, FAO/WHO allergenicity assessment rules were applied, as well as procedures suggested by König et al. (2004) = 80 aa sliding window search and 8 aa epitopes = 0 for all three. Protein toxicity: assayed by amino acid sequence search with sequences deposited in in Genbank = 0 Stability, digestibility TO DO Mouse oral gavage study TO DO

25 Safety to the environment Environment: No wild relatives in Africa Gene flow var to var? Infrequent successful out-crossing Germination difficult Fruit not attractive Vine multiplication Improved varieties Gene flow in sweepotato is not an issue. Andersson and de Vicente, Gene flow between crops and their wild relatives. Johns Hopkins University Press, Baltimore, Maryland, USA

26 Safety to the environment A review of non-target organism present in sweetpotato cropping system. List of arthropods present in SP production system (literature, phytosanitary services, on-going GM deregulation, experts opinion) Coleopteran, other orders Charismatic: honey bee, ladybird Only ladybird, ground, and rove beetles as coleopteran predators might need further investigation.

27 Capacity building in infrastructure Contribution to the establishment of the NaCRRI Biosciences Facility Sweetpotato BS level 2 S/house and Confined Field Trial site

28 Capacity building in human All African scientists have completed the e-distance learning postgraduate course Biosafety in Plant Biotechnology at the Ghent University (Be).

29 Knowledge sharing communication 1 st Science and Communication workshop on Biotech sweetpotato held on August 9-12 at ILRI Campus, Kenya: Trained RP2 scientist to communicate better Review of the project Pamphlets:

30 Bt as B ig T hanks to: [R.O.M. Mwanga, W.J. Moar, M. Ghislain]*, D. Delmer, Z. Dapeng, B. Odongo, M. van Montagu, L. Gheysen, J. Kreuze, G. Hareau, R. Labarta, B. Kiiza, G. Ssemakula, J. Machuka, J. Tovar, K. Prentice, M. Ormachea, C. Rivera, S. Manrique, L. Wamalwa, M. Ekobu, M. Solera, A. Sefasi, R. Rukarwa, M. Gati [Namulonge Agricultural Research Organization, Auburn University, International Potato Center]*; and other institutions who joined the initiative: University of Ghent (IPBO), Makerere University, Kenyatta University, BecA / ILRI, DDPSC, UPRM, Without forgetting our sponsors: Rockefeller Foundation, USAID, DGDC (Belgium); B&M GATES Foundation *Triumvirates