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 www.iita.org A member of CGIAR consortium
Agriculture Innovations • Nearly a billion people going hungry in the world everyday. • Agricultural output needs to be doubled to feed the world population in 2050. • 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. www.iita.org A member of CGIAR consortium
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. www.iita.org A member of CGIAR consortium
Global Area of Biotech Crops • In 20 years, GM crops have almost 100-fold increase since commercialization. www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
Transgenic Banana for Africa • Disease resistance - Bacterial wilt - Black sigatoka - Fusarium wilt - BBTV • Pest resistance - Nematodes • Biofortification - Pro-vitamin A and iron www.iita.org A member of CGIAR consortium
Transformation System for Banana & Plantain • Robust transformation system using cell suspensions • Gonja Manjaya , Gros Michael, Williams Cavendish, Sukali Ndiizi Tripathi et al . 2012 In Vitro Cell Dev Biol- Plant Tripathi et al . 2015 Frontiers in Plant Science www.iita.org A member of CGIAR consortium
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 2001. • The disease has also been reported in east Africa. Tripathi et al. 2009, Plant Disease www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
Control of BXW • Uprooting of diseased plants • 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 insect- mediated transmission Atim et al. 2013 Plant Disease Adikini et al 2013 Can J Plant Pathol Blomme et a l. 2014 Eur J Plant Pathol www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
Generation of Transgenic Plants • Over expression of sweet pepper Hrap or Pflp in transgenic banana. • Sukali Ndiizi, Nakinyika, Gonja Manjaya. Tripathi et al . 2010 Mol. Plant Pathology Namukwaya et al . 2012 Transgenic Research Tripathi et al. 2010 Acta Hort. www.iita.org A member of CGIAR consortium
Evaluation of Transgenic Plants • 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 Tripathi et al . 2010 Mol Plant Pathol Namukwaya et al . 2012 Transgenic Research Tripathi et al 2007 Eur. J Plant Pathology Adikini et al . 2011 Plant Pathology www.iita.org A member of CGIAR consortium
Molecular Characterization M P 1 2 3 4 5 6 7 8 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 . 2010 Mol Plant Pathol Northern blot analysis of transgenic plants Namukwaya et al . 2012 Transgenic Research www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
Evaluation for BXW Resistance www.iita.org A member of CGIAR consortium
Disease Evaluation www.iita.org A member of CGIAR consortium
Second ratoon crop www.iita.org A member of CGIAR consortium
External BXW Symptoms on Fruits www.iita.org A member of CGIAR consortium
Internal BXW Symtoms www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
Agronomic Performance of Best Transgenic Lines www.iita.org A member of CGIAR consortium
Yield Parameters of Best Transgenic Lines www.iita.org A member of CGIAR consortium
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. www.iita.org A member of CGIAR consortium
Product Development • 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 2016. • BXW resistant bananas will be ready for release in 2021. www.iita.org A member of CGIAR consortium
Biosafety of PFLP and HRAP • 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. www.iita.org A member of CGIAR consortium
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. www.iita.org A member of CGIAR consortium
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 www.iita.org A member of CGIAR consortium
Resistance Technologies • Maize cystatin that inhibits nematode digestive cysteine proteinases • Synthetic peptide that disrupts nematode chemoreception www.iita.org A member of CGIAR consortium
Generating Transgenic Plantain • Plantains transformed – Cystatin – Peptide – Additive • Generated about 250 transgenic lines of Gonja manjaya • Confirmed by – PCR – Cystatin • Western – Peptide M - + lines • Bioassay 1 _ PCR – Nematode challenge in Kb 0.5_ screenhouse 20 _ Western Kda Roderick et al. 2012 Mol Plant Pathol www.iita.org A member of CGIAR consortium
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