Application of Genetic Modification for Crop Improvement: Case Studies from African Crops

Transcription

1 Application of Genetic Modification for Crop Improvement: Case Studies from African Crops Leena Tripathi International Institute of Tropical Agriculture, Nairobi, Kenya 5 th Annual Biosafety Conference, Nairobi, 17 th August 2016

2 Agriculture Innovations Nearly a billion people going hungry in the world everyday. Agricultural output needs to be doubled to feed the world population in World can only meet its future food needs through harnessing scientific innovation and technology in agriculture. It's also critical to meet the global challenges of producing more food with less land and water, improving nutrition, and helping farmers adapt to climate change.

3 Genetic Engineering Potential to transform agriculture in Africa. To date, only three African countries (South Africa, Burkina Faso and Sudan) have adopted GM crops. Other countries such as Uganda, Kenya, Nigeria, Cameroon, Malawi and Ghana are at the forefront of leveraging biotechnology to address local agricultural challenges. They are focusing on problems such as pests, disease, drought and low nutritional content of staple crops.

4 Global Area of Biotech Crops In 20 years, GM crops have almost 100-fold increase since commercialization.

5 Staple Crops for Africa Most of the staple crops in Africa receive little attention from the private sector. Banana and Plantain Cassava Cowpea Yam Enset

6 Banana and Plantain Rank 4 th most important crop in Africa. World Musa production is 140 million tonnes. A third of the bananas produced globally are grown in Africa. East Africa is the largest banana producing and consuming region in Africa. Uganda is the world s second largest producer. Production is threatened by various constraints pests like nematodes and weevils diseases like fungal, bacterial and viral

7 Transgenic Banana for Africa Disease resistance - Bacterial wilt - Black sigatoka - Fusarium wilt - BBTV Pest resistance - Nematodes Biofortification - Pro-vitamin A and iron

8 Transformation System for Banana & Plantain Robust transformation system using cell suspensions Gonja Manjaya, Gros Michael, Williams Cavendish, Sukali Ndiizi Tripathi et al In Vitro Cell Dev Biol- Plant Tripathi et al Frontiers in Plant Science

9 Banana Xanthomonas wilt (BXW) BXW caused by Xanthomonas campestris pv. musacearum. First reported more than 45 years ago in Ethiopia on enset and then on banana. Outside of Ethiopia, it was reported in Uganda in The disease has also been reported in east Africa. Tripathi et al. 2009, Plant Disease

10 Banana Xanthomonas Wilt The impacts of BXW are both extreme and rapid. All the varieties are susceptible. Overall economic losses were estimated at $2-8 billion over a decade. Tripathi et al. 2009, Plant Disease

11 Xanthomonas Wilt Disease: Spread Insect vectors Infected planting materials Contaminated farming tools Traded banana products Biruma et al. 2007, Af. J. Biotech. Tripathi et al. 2009, Plant Disease

12 Uprooting of diseased plants Control of BXW Chemical destruction of diseased mats Use of clean farming tools Debudding of male buds Use of clean planting materials Improving soil fertility Use of varieties that escape insectmediated transmission Atim et al Plant Disease Adikini et al 2013 Can J Plant Pathol Blomme et al Eur J Plant Pathol

13 Transgenic Technology Resistance has been the best and most cost-effective method of managing bacterial diseases. Pflp (plant ferredoxin-like protein) and Hrap (HR assisting protein) are cloned from sweet pepper, Capsicum annuum. Intensify the HR caused by harpin (a proteineous elicitor secreted from bacterial pathogen). Elicitor-induced resistance is not specific against particular pathogens. USAID Project: IITA, NARO, AATF, KALRO

14 Over expression of sweet pepper Hrap or Pflp in transgenic banana. Sukali Ndiizi, Nakinyika, Gonja Manjaya. Generation of Transgenic Plants Tripathi et al Mol. Plant Pathology Namukwaya et al Transgenic Research Tripathi et al Acta Hort.

15 All the PCR +ve lines were evaluated Using in vitro plants Potted plants in screen house No bacteria was found at the point of inoculation in healthy transgenic lines after 6 weeks PCR Selective medium Evaluation of Transgenic Plants Tripathi et al Mol Plant Pathol Namukwaya et al Transgenic Research Tripathi et al 2007 Eur. J Plant Pathology Adikini et al Plant Pathology

16 Molecular Characterization M P C Southern blot analysis of transgenic plants RT-PCR analysis of transgenic plants using gene specific primers; Western blot analysis of transgenic plants Tripathi et al Mol Plant Pathol Namukwaya et al Transgenic Research Northern blot analysis of transgenic plants

17 Confined Field Trial 65 transgenic lines (45 lines with Hrap gene and 20 lines with Pflp gene) were planted in CFT BXW disease evaluation Agronomic performance Nature News 2010; Nature Biotech News 2011

18 Evaluation for BXW Resistance

19 Disease Evaluation

20 Second ratoon crop

21 External BXW Symptoms on Fruits

22 Internal BXW Symtoms

23 BXW Resistant lines 11 lines (7 Hrap lines and 4 Pflp lines) showed 100% resistance in field for 3 generations. Tripathi et al. 2014, Nature Biotechnology

24 Agronomic Performance of Best Transgenic Lines

25 Yield Parameters of Best Transgenic Lines

26 2 nd Confined Field Trial 10 lines (7 Hrap and 3 Pflp) with 12 replicates were further evaluated in 2 nd CFT in Uganda from Sept 2013-Dec 2015.

27 Durable and enhance resistance Gene pyramiding Transformation 2 dessert varieties for Kenya 2 matooke varieties in Uganda Transgenic bananas with stacked genes for durable resistance to BXW Hundreds of lines developed Tested in glasshouse trial CFT in Kenya and Uganda with stacked lines in BXW resistant bananas will be ready for release in Product Development

28 Bioinformatics sequence comparisons were performed comparing the amino acid sequences of both proteins to known allergens and toxins (AllergenOnline.org and NCBI). No convincing evidence was found to suggest that the HRAP or PFLP proteins represent risks of allergy or toxicity to humans. Peppers are commonly consumed with rare cases of allergy and no reports of toxicity. Food safety studies will be done. Nutritional quality will be checked. Biosafety of PFLP and HRAP

29 Non-target Effect of PFLP and HRAP The expression of Pflp and Hrap genes did not show any effect on non-target microorgansms in soil.

30 Nematodes Nematode losses 30-50% yield to banana in Africa. Limited sources of nematode resistance present in the Musa gene pool. Some resistance has been identified against Radopholus similis, but this needs to be combined with consumer-acceptable traits. Several species occur in the same soils Radopholus similis, Pratylenchus spp, Helicotylenchus spp, Meloidogyne spp Gene stacking the best way forward Several approaches available Partners: IITA, University of Leeds, NARO

31 Resistance Technologies Maize cystatin that inhibits nematode digestive cysteine proteinases Synthetic peptide that disrupts nematode chemoreception

32 Western PCR Generating Transgenic Plantain Plantains transformed Cystatin Peptide Additive Generated about 250 transgenic lines of Gonja manjaya Confirmed by PCR Cystatin Western Peptide Bioassay Nematode challenge in screenhouse Roderick et al Mol Plant Pathol 1 _ Kb 0.5_ 20 _ Kda M - + lines

33 Glasshouse Trials Establishing the trial Harvest Roderick et al Mol Plant Pathol

34 Confined Field Trial Twelve promising lines were planted in CFT in Uganda for further evaluation.

35 % Resistance to nematodes Nematode Resistance 100% harvest ** *** *** *** *** *** *** *** *** *** *** *** flowering * ** *** ** ** *** *** *** *** *** growth * ** ** ** *** ** *** *** 80% 60% 40% 20% 0% +nem D12 D30 D46 C15 P53 P46 C6 P78 D66 D14 P48 P77 Tripathi et al Scientific Reports

36 Precision Delivery of Transgene Root-cap-specific promoter identified for Arabidopsis (gene MDK4-20). Maize root cap promoter (ZmRCP1) homologue identified and cloned. Tested the construct in banana using reporter gene. It can be used for delivery of the peptide to the rhizosphere via root border cells. Onyango et al. (2016) J Biol Res

37 BBTV is a major limiting factor in banana production. No known sources of resistance to BBTV within banana germplasm. No opportunity to incorporate BBTV resistance into any of the conventional banana breeding programs. Developing transgenic bananas using RNAi targeting - BBTV Aphids Banana Bunchy Top Virus BMGF Project- QUT, IITA and DARS

38 Genetic transformation of African farmer preferred cultivars of cassava Using friable embryogenic callus 7 east African cultivars 5 west African cultivars Cassava Transformation Platform Developing resistance CBSD CBB Nyaboga et al Frontiers in Plant Biotech Nyaboga et al Frontiers in Plant Biotech

39 Genetic Transformation of Yam Agrobacterium-mediated transformation system established for yam using apical meristems. It takes 5 6 months from transformation to regeneration of complete transgenic plant. A D E F A member of of the CGIAR consortium Nyaboga et al Frontiers in Plant Sci

40 Yams are susceptible to a number of species of endoparasitic nematodes. Yam nematodes reproduce and build up large populations in stored tubers, causing severe damage. Nematodes also facilitate fungal and bacterial attacks that cause anthracnose disease, dry rot, soft rot and wet rot. Nematode Resistant Yam Technologies proven in plantain can be applied to yam.

41 Enset is staple food for more than 20 million people in Ethiopia. Agrobacteriummediated transformation system established. Genetic Transformation of Enset

42 Enset production has been severely threatened by bacterial wilt disease. Control of Bacterial Wilt in Enset The technology (Pflp and Hrap) mastered in the proof-of-concept for control of BXW can be extended to enset. BMGF Project- IITA and EIAR.

43 Conclusions GM approaches shows enormous potential for development of varieties resistant to diseases & pests. The techniques mastered in these proof-of-concept states can be extended to a wide range of other crops. Technology for nematode resistance can be applied to yams BXW resistance technology can be applied to bacterial diseases of enset, cassava etc. There are several products on horizon for Africa. BXW resistant banana planned to be commercialized in 2021.

44 Partners NARO-Uganda EIAR- Ethiopia KALRO- Kenya University of Leeds, UK Donald Danforth Plant Science Centre Qeensland University of Technology, Australia ETH AATF Academia Sinica, Taiwan Acknowledgements Financial Support Bill & Melinda Gates Foundation CGIAR Research Program DFID/BBSRC Gatsby Charitable Foundation USAID NSF