Cassava improvement in the era of “agrigenomics”

Ismail Yusuf Rabbi (i.rabbi@cgiar.org), Melaku Gedil, Morag Ferguson, and Peter Kulakow
I. Rabbi, Postdoctoral Fellow (Molecular Genetics); M. Gedil, Head, Bioscience Center, IITA, Ibadan, Nigeria; M. Ferguson, Molecular Geneticist, IITA, Nairobi, Kenya; and P. Kulakow, Cassava Breeder, IITA, Ibadan, Nigeria

Pro-vitamin A 'yellow root' cassava developed by the IITA cassava breeding program. Photo by IITA.
Pro-vitamin A 'yellow root' cassava developed by the IITA cassava breeding program. Photo by IITA.

In the last 45 years, IITA has played a pivotal role in the genetic improvement of cassava for resource-poor farmers in sub-Saharan Africa (SSA). More than 400 cassava varieties have been developed that are not only high yielding but also resistant to diseases and pests. Many of these improved varieties have been extensively deployed in SSA and have helped to avert humanitarian crises caused by the viral disease pandemics that devastated local landraces in East and Central Africa. The cassava breeding program in Ibadan has a collection of more than 750 elite cassava clones representing current and historical materials accumulated over the last 45 years. These materials, referred to as the genetic gain collection (GGC), are accompanied by extensive field evaluation (phenotypic) data. In addition, the active breeding collection contains over 1000 African landraces and more than 400 new advanced breeding clones that are also accompanied by phenotypic data, including observations of disease and pest resistance, plant architecture, flowering ability, and performance in storage root yield. The most recent success of the conventional cassava breeding program culminated in the release of three vitamin A cassava varieties by the Government of Nigeria. These varieties (IITA TMS I011368, IITA TMS I011371, and IITA TMS I011412) were first cloned from seedlings in Ibadan in 2001 and have been subjected to extensive field testing throughout Nigeria. While almost all cassava in Nigeria are currently white fleshed, vitamin A cassava produces yellow-fleshed roots with nutritionally significant concentrations of carotenoids that produce vitamin A in the human body when consumed as yellow gari or fufu. In cooperation with HarvestPlus, IITA and partners will distribute vitamin A cassava planting materials to more than 25,000 farmers in 2013. New yellow-fleshed genotypes in the pipeline promise continued improvement in pro-vitamin A content, yield, and dry matter in the coming years.

Preparation of cassava DNA for genotyping by sequencing. Photo by IITA.
Preparation of cassava DNA for genotyping by sequencing. Photo by IITA.

As the vitamin A cassava illustrates, the genetic improvement of cassava has mostly been achieved through conventional breeding methods based on phenotypic selection. The only known direct application of molecular markers in cassava breeding is selection for resistance to cassava mosaic disease and cassava green mite. Recent advances and a reduction in the cost of the next-generation sequencing technologies now promise to usher in a new era for cassava breeding that will combine the success of conventional hybridization, selection, and multilocational yield trials with the latest advances in genomic resources.

Setting the stage for “next-generation cassava breeding”
Cognizant of the potential of marker technologies to improve the efficiency and effectiveness of cassava breeding, IITA, in collaboration with partners, embarked on the development and deployment of molecular markers1. With the recent accumulation of genomic resources in cassava research, including the first full cassava genome sequence2, our emphasis at IITA has shifted towards the application of these resources in molecular breeding3. One recent achievement is the identification and validation of nearly 1500 single nucleotide polymorphism (SNP) markers through an international collaboration led by IITA’s geneticist, Morag Ferguson4. These SNPs have been converted to a highly parallel hybridization-based genotyping system that has been shared with the international cassava research community through partnership with the Generation Challenge Program (GCP).

An example of an SNP genotyping data plotted with KBioscience’s SNPviewer software. Inset: raw SNP genotyping data from Illumina’s GoldenGate®assay.
An example of an SNP genotyping data plotted with KBioscience’s SNPviewer software. Inset: raw SNP genotyping data from Illumina’s GoldenGate®assay.

In addition, the first SNP-based genetic linkage map of cassava has been developed by IITA in collaboration with Heneriko Kulembeka of the Agricultural Research Institute (ARI), Ukiriguru, Tanzania. A linkage map is analogous to landmarks (SNP markers in this case) placed along chromosomes that guide researchers to genes or genomic regions controlling traits of interest. Such a linkage map is an indispensable tool for marker-assisted selection (MAS). SNP and SSR markers have also been applied to uncover quantitative trait loci (QTL) associated with resistance to cassava brown streak disease (CBSD)―which is ravaging cassava production in Eastern and Southern Africa―in a collaboration between IITA, CIAT, and ARI-Tanzania. Another dramatic development in cassava genomics is the recently completed sequencing of the cassava genome through the partnership of the US Department of Energy’s Joint Genome Institute and 454 Life Sciences2.

Genotyping-by-sequencing
The progress in next- generation technologies has drastically reduced the costs of DNA sequencing so that genotyping-by-sequencing (GBS) is now feasible for species such as cassava, ushering in a new era of agricultural genomics5. This will revolutionize the application of genomic tools for cassava improvement. GBS involves the cutting of genomic DNA into short pieces at specific locations using a restriction enzyme. The ends of these pieces are sequenced using techniques that allow sequencing of many samples at the same time. The beauty of this method is the use of adaptors containing barcodes (unique tags) that are enzymatically joined to the digested DNA fragments, enabling simultaneous sequencing or multiplexing of up to 384 samples in one sequencing reaction. This economy of scale greatly reduces the cost of processing each individual DNA to less than $10/sample. Approximately 200,000 markers can be identified and mapped in a very short time. With this powerful tool, breeders may conduct genomics-based research that was inconceivable a couple of years ago. Some of the exciting new research applications include polymorphism discovery, high-density genotyping for QTL detection and fine mapping, genome-wide association studies, genomic selection, improving reference genome assembly, and kinship estimation.

High-density QTL mapping and fine mapping
In the past, a limitation for QTL mapping was the number of markers on a genetic linkage map. With new SNP-based technologies this is no longer a limitation. This allows for fine mapping of QTLs so long as a sufficient number of individuals in the mapping population can be developed. IITA, in collaboration with national partners [ARI-Tanzania and National Crops Resources Research Institute (NaCRRI), Uganda], is using SNPs to discover QTLs associated with sources of tolerance for CBSD.

Preparation of gari, the most popular food product from cassava. Photo by IITA.
Preparation of gari, the most popular food product from cassava. Photo by IITA.

The next frontier for cassava genomics
Using the genotyping by sequencing approach, scientists from IITA and Cornell University, USA, are currently genotyping more than 2000 accessions of cassava, including released varieties, advanced breeding lines, and landraces from Africa. This is a pilot study of genomic selection funded by the Bill & Melinda Gates Foundation to explore the potential for using the IITA breeding collection, including genetic gain, local germplasm, and current advanced breeding lines, as the base population to begin genomic selection for West Africa. The IITA breeding collection has been extensively characterized in many locations and over many years. The convergence of high-density SNP data and extensive phenotypic data in IITA’s cassava collection sets the stage for the implementation of genome-wide association studies (GWAS) and genomic selection (GS) in breeding. The aim of GWAS is to pinpoint the genetic polymorphisms underlying agriculturally important traits. In GWAS, the whole genome is scanned for significant marker-trait associations, using a sample of individuals from the germplasm collections, such as a breeder’s collection. This approach of “allele mining” overcomes the limitations of traditional gene mapping by (a) providing higher resolution, (b) uncovering more genetic variants from broad germplasm, and most importantly, (c) creating the possibility of exploiting historical phenotypic data for future advances in breeding cassava.

A schema of genomic selection (GS) processes, starting from phenotyping and genotyping of the training population and selection of parental candidates via genomic estimated breeding value (GEBV)–based selection. Note that selection model improvement can be performed iteratively as new penotype and marker data accumulate.
A schema of genomic selection (GS) processes, starting from phenotyping and genotyping of the training population and selection of parental candidates via genomic estimated breeding value (GEBV)–based selection. Note that selection model improvement can be performed iteratively as new penotype and marker data accumulate.

GS is a breeding strategy that seeks to predict phenotypes from high-density genotypic data alone, using a statistical model based on both phenotypic and genotypic information from a “training population”. For cassava, phenotyping is the slowest and most expensive phase of the crop’s breeding cycle because of the crop’s low multiplication ratio of between 5 and 10 cuttings/plant. Thus, it takes several cycles of propagation (up to 6 years) to carry out a proper multilocational field trial evaluation. The implementation of GS at the seedling stage should: (a) dramatically reduce the length of the breeding cycle, (b) increase the number/unit time of crosses and selections, and (c) increase the number of seedlings that could be accurately evaluated. The reduced breeding cycle means that the ”engine of evolution,” i.e., recombination and selection, can proceed at a rate that is three times as fast as phenotypic-based selection, while saving resources. In conclusion, cassava breeding in IITA is being redefined, thanks to the increasing availability and deployment of genomic resources. Combining these resources with IITA’s long-standing conventional breeding pipeline means that the best days of cassava improvement lie ahead. These efforts will ultimately satisfy the increasing need for more healthy and nutritious food produced in environmentally sustainable ways.

References
1 Lokko et al. 2007. Cassava. In: Kole et al (ed). Genome mapping and molecular breeding in plants, Vol. 3. Pulses, Sugar and Tuber Crops. Springer-Verlag Berlin Heidelberg.
2 Prochnik S., P.R. Marri,B. Desany, P.D. Rabinowicz, et al. 2011. Tropical Plant Biol. doi:10.1007/s12042-011-9088-z. 3 Ferguson M., I.Y. Rabbi, D-J.Kim, M. Gedil, L.A.B. Lopez-Lavalle, and E. Okogbenin. 2011a. Tropical Plant Biol. DOI 10.1007/s12042-011-9087-0.
4 Ferguson M.E., S.J. Hearne, T.J. Close, S. Wanamaker, W.A. Moskal, C.D. Town, J. de Young, P.R. Marri, I.Y. Rabbi, and E.P. de Villiers. 2011b. Theor Appl Genet. DOI: 10.1007/s00122-011-1739-9.
5 Elshire R., J. Glaubitz, Q. Sun, J. Poland, and K. Kawamoto. 2011. PLoS ONE 6:e19379.

Genomics for transforming yam breeding

Ranjana Bhattacharjee (r.bhattacharjee@cgiar.org), Melaku Gedil, and Antonio Lopez-Montes
R. Bhattacharjee, Molecular Geneticist; M. Gedil, Head, Bioscience Center; A. Lopez-Montes, Yam Breeder, IITA, Ibadan, Nigeria

Breeding challenges in yam
Yam (Dioscorea spp.), a multi-species, polyploidy, and vegetatively propagated crop, is an economically important staple food for more than 300 million people in West Africa, Asia, Oceania, and the Caribbean. The five major yam-producing countries in West Africa (Bénin, Côte d’Ivoire, Ghana, Nigeria, and Togo) account for 93% of worldwide production. Dioscorea rotundata and D. alata are the species most commonly cultivated in West Africa1.

Scientists strategizing genomics for precision breeding. Photo by L. Kumar.
Scientists strategizing genomics for precision breeding. Photo by L. Kumar.
The genetic improvement of yam is faced with several constraints, including the long growth cycle (about 8 months or more), dioecy, plants that flower poorly or not at all, polyploidy, vegetative propagation, heterozygous genetic background, and poor knowledge about the genetics of the crop2. Progress has been made in breeding to develop F1 full-sib mapping populations from crossing male and female parents of D. rotundata for traits such as multiple tuber production, improved cooking quality, and virus disease resistance; and of D. alata for resistance to anthracnose, improved cooking quality, and reduced tuber oxidation3. These are valuable sources of populations for genetic analysis in yam for its improvement.

Current status of yam genomics
There is no convenient model system for yam genomics. In recent years, some progress has been made in the development of molecular markers to assess their potential for germplasm characterization and phylogenetic studies in D. rotundata-cayenensis and their wild progenitors, such as D. abyssinica and D. prahensilis. Two framework linkage maps were constructed using D. alata that included 338 AFLP markers on 20 linkage groups with a total map length of 1055 cM; and D. rotundata in which 107 AFLP markers were mapped on 12 linkage groups (585 cM) for the male and 13 linkage groups (700 cM) for the female. Three quantitative trait loci (QTLs) on the male and one QTL on the female were identified for resistance to yam mosaic virus (YMV). Similarly, one AFLP marker was found to be associated with anthracnose resistance on linkage group 2, explaining about 10% of the total phenotypic variance.

Another linkage map was generated for D. alata based on 508 AFLP markers that covered a total length of 1233 cM on 20 linkage groups, accounting for about 65% of the entire genome. Genes conferring resistance to YMV have been identified in D. rotundata and to anthracnose in D. alata by the successful use of bulked segregant analysis (BSA). Two RAPD markers, OPW18850 and OPX15850, closely linked in coupling phase with the dominant YMV-resistance locus Ymv-1 were identified. Similarly, two RAPD markers, OPI171700 and OPE6950, closely linked in coupling phase with anthracnose resistance gene, Dcg-1, were identified2.

Designing molecular markers using a bioinformatics platform. Photo by A. Alonge, IITA.
Designing molecular markers using a bioinformatics platform. Photo by A. Alonge, IITA.
Enriching the repertoire of molecular markers
In an effort to develop additional genomics resources, IITA was involved in sequencing ESTs from a cDNA library constructed from floral tissue. However, the first several hundred sequences were predominantly housekeeping genes. Recently, in a collaborative project with University of Virginia through USAID-Linkage funds, several thousand ESTs were generated using cDNA libraries from yam leaf tissues challenged with Colletotrichum gloeosporioides, the fungal pathogen responsible for yam anthracnose disease. This resulted in the identification of >800,000 EST sequences, from which about 1152 EST-SSRs were generated in D. alata for use in a yam improvement program. Although AFLP markers have been used for generating linkage maps so far, efforts are under way to saturate the maps with these EST-SSRs to identify the genomic regions associated with resistance to anthracnose disease.

DNA barcoding
Species identification in the genus Dioscorea has remained a challenge when active domestication is continuing in several parts of West Africa. Research on DNA barcoding is under way using chloroplast markers (rbcL, matK, and trnH-psbA) to understand the phylogenetic relationship between different species and also to get an insight into the ongoing domestication process.

Whole genome sequencing
Important considerations for the whole genome sequencing of yam include the genome size, ploidy level, and availability of homozygous clones. Estimation of the genome sizes of various Dioscorea species showed widely variable figures: D. alata and D. rotundata have genome sizes of about 800 mega base pairs (Mbp). Recently, an initiative was launched at IITA in collaboration with the Japan International Research Center for Agricultural Sciences (JIRCAS) to complete the whole genome sequencing of D. rotundata. Preliminary data yielded reasonable sequences. Further work is in progress to generate additional sequence data from the BAC library to facilitate the assembly of the genome which will culminate in producing the first draft genome sequence of Dioscorea species. Additional genomic information produced by resequencing several breeding materials and a parallel project in transcriptome analysis are poised to result in the discovery of a large number of molecular markers and help in the annotation of the genome.

Transcriptome analysis
In contrast to the genome sequence, which is fixed and uniform in all cells of a particular organism, transcriptome refers to the study of the total set of transcripts (expressed genes) in a given cell/tissue at a particular developmental stage or external environmental condition that could influence the physiology of the cell/tissue. IITA, in collaboration with USDA-Agricultural Research Service, Stoneville, embarked on RNAseq, the latest revolutionary tool for transcriptome profiling, based on differential gene expression for anthracnose disease. One of the expected outcomes of this project is to enrich the genomic resources available for yam improvement, including the discovery of SNPs. The latest informatics and statistical methods will be applied to saturate the available linkage map and high resolution mapping of the QTL(s) for anthracnose resistance in different genetic backgrounds.

Genotyping-by-sequencing
With advances in the next generation technologies, the costs of DNA sequencing have come down to such an extent that genotyping-by-sequencing (GBS) is now possible in almost all crops. IITA has recognized the potential of such innovative techniques in accelerating the breeding of clonally propagated crops, such as yam. Hence, in an ongoing USAID-Linkage project, a diverse panel of D. alata genotypes, including parents of available mapping population progenies segregating for anthracnose disease will be genotyped by sequencing to identify a large set of SNPs and determine the divergence among the parents.

Yam roots. Photo by IITA.
Yam roots. Photo by IITA.
Conclusions
To meet the steadily increasing demand, the viable approach is to adopt the innovative plant breeding strategies for yam that integrate the latest innovations in molecular technologies with conventional breeding practices. As efforts are under way to obtain the complete genome sequences and the development of additional genomic resources, the groundwork for deploying yam molecular breeding has been laid. With the availability of new genomic markers and GBS, it would be possible to fingerprint yam germplasm to identify duplicates/mislabeled accessions, to conduct diversity analysis and association mapping. As the genus Dioscorea contains several other useful species, comparative genomic tools can be used to transfer or deduce genetic and genomic information in other species.

References
1 Gedil, M. and A. Sartie. 2010. Aspects of Applied Biology 96:123–135.
2 Mignouna, H.D., M.M. Abang, and R. Asiedu. 2007. Yams. Pages 271–296 in: Genome mapping and molecular breeding Vol. 3: Pulses, Sugar and Tuber Crops, edited by C. Kole. Springer, Heidelberg, Berlin, New York, and Tokyo.
3 Sartie, A. and R. Asiedu. 2011. Development of mapping populations for genetic analysis in yams (Dioscorea rotundata Poir. and Dioscorea alata L.). African Journal of Biotechnology 10: 3040–3050.

Leveraging “agrigenomics” for crop improvement

Melaku Gedil (m.gedil@cgiar.org) and Ismail Rabbi
M. Gedil, Head, Bioscience Center; I. Rabbi, Postdoctoral Fellow (Molecular Genetics), IITA, Ibadan, Nigeria

Harnessing state-of-the art genomics technologies
The potential application of “Omics” technology, as demonstrated by the steadily growing impact of biosciences, in alleviating the multitude of constraints in agricultural production is rapidly becoming a reality with the advent of next-generation DNA sequencing and genotyping technologies, high throughput (HTP) metabolomics and transcriptomics, informatics, and decision-making tools. These technologies, together with rapidly evolving bio-computational tools, are accelerating the discovery of genes and closely linked molecular markers underlying important traits, leading to the rapid accumulation of genomic resources necessary for devising an efficient and effective breeding strategy geared toward the faster development of varieties of choice.

Researchers in IITA's Bioscience Center. Photo by L. Kumar.
Researchers in IITA's Bioscience Center. Photo by L. Kumar.
The state-of-the-art technologies including the next-generation sequencing (NGS) for genome and transcriptome analysis, as well as genotyping-by-sequencing (GBS) are being adopted in R4D programs at IITA. For instance, the NGS through outsourcing and multi-partner collaboration; the RNAseq for HTP expression study in cassava; the Illumina’s Golden Gate Assay for HTP single nucleotide polymorphism (SNP) genotyping in cassava, soybean, and maize as well as GBS in maize and cassava. Data generated by these techniques are being applied for marker-assisted recurrent selection (MARS) of drought-tolerant maize, and genome selection (GS) for high-yielding, disease-resistant cassava.

Development of an integrated molecular breeding platform
The new technologies, however, are very data-intensive and demand advanced computational and communication technologies and infrastructure for data acquisition, analysis, and management. For the effective integration of genomics technologies in our breeding schemes, we are building capacity (connectivity to the internet, the necessary hardware/software, and skilled personpower) to acquire, store, and analyze terabytes of data.

The Generation Challenge Program (GCP) of the CGIAR is developing an integrated breeding platform (IBP) to build a comprehensive and integrated crop information system enabling linkages among molecular, phenotypic, and pedigree data. The maize version of International Crop Information System (ICIS), dubbed International Maize Information System (IMIS), has been expanded to include all pedigrees of IITA maize under the Drought Tolerant Maize for Africa (DTMA) project. It has some functionality in terms of molecular data storage but this is limited and we are now generating data sets of hundreds of thousands of markers per line that require different storage solutions. The GCP is consulting with other initiatives such as iPlant and DArT and is working on collaboratively creating solutions for the needs of several user-cases including DTMA, Tropical Legumes (TL)-I, and TL-II projects. In the IBP initiative, IITA is the leading crop center to host the main web-accessible databases of cassava, cowpea, yam, and soybean. The form and functionality of the databases are still a work in progress although activities are ongoing in the application of current versions of ICIS to cassava, yam, and cowpea.

In view of the IBP initiative, we are developing a bioinformatics capacity to (a) manage the newly generated genomic resources of IITA’s research crops, particularly those clonally propagated, (b) use the genomic resources in the public sector for soybean and maize, (c) use comparative genomics techniques for other African orphan crops of high importance, such as cassava, yam, and cowpea, and (d) create a bioinformatics center of excellence to train and provide access for African research scientists.

HTP by genotyping and informatics support tools
The increasing affordability of the NGS technologies has shifted critical consideration from genotyping to phenotyping. According to leading experts, it is now cheaper to genotype than to phenotype a plant. Quality phenotypic data are essential for the interpretation and use of the deluge of genomic data to identify the changes in DNA sequences that influence important traits. The fact that priority agronomic traits are complex and polygenic and interact with the environment necessitates conducting extensive and precise multi-environment evaluations of candidate breeding materials (over several years and in several locations). Therefore, there is a need to invest in precision phenotyping of traits and data capture (from electronic sample tracking to non-invasive HTP) through the use of hand-held devices such as barcode readers and near-infrared spectroscopy. Efforts are being made to develop rapid and accurate phenotyping protocols to integrate with genomic tools in establishing breeding schemes at IITA.

A wide array of techniques and tools is being deployed to associate molecular markers with desirable phenotypic traits. Associated markers can be used to accelerate germplasm enhancement via MARS, marker-assisted backcrossing for the introgression of disease resistance and other simple traits, hence bypassing the necessity of evaluating breeding materials in the field; MARS for rapid cycle population improvement in bi-parental crosses based on genomic estimated breeding value; and GS based on a model developed with a training population to select untested samples.

Our efforts to harness the unparalleled scientific progress in the fields of genomics and bioinformatics are expected to find solutions to the recalcitrant problems confronting small-holder farmers in sub-Saharan Africa.

Transgenics in crop improvement research

Leena Tripathi (l.tripathi@cgiar.org)
Biotechnologist, IITA, Nairobi, Kenya

Biotechnology has opened unprecedented avenues for exploring biological systems. Transgenics is one of the key techniques particularly useful for the genetic improvement of crops that are not amenable to conventional breeding, such as those that are vegetatively propagated. In IITA, transgenic technologies are being used for improving banana/plantain (Musa sp.), cassava (Manihot esculenta), and yam (Dioscorea sp.).

Harvested bunch of transgenic banana, Kampala, Uganda. Photo by L. Tripathi.
Harvested bunch of transgenic banana, Kampala, Uganda. Photo by L. Tripathi.
Genetic transformation platform
An efficient protocol for plant regeneration and transformation is a prerequisite for the successful use of transgenic technologies. Despite the technical difficulties in transforming monocot species, efficient transformation protocols that are embryogenic cell suspension based and Agrobacterium mediated have been established for many cultivars of banana/plantain. This system, however, is a lengthy process and cultivar dependent. Therefore, a transformation protocol using meristematic tissues was also established which is rapid and genotype independent. These protocols have paved a way for the genetic manipulation of banana/plantain by incorporating agronomically important traits such as those conferring resistance to diseases or pests as well as tolerance to abiotic stress factors.

Agrobacterium-mediated transformation protocols for three popular cassava varieties preferred by African farmers were established through somatic embryogenesis. A regeneration and transformation protocol is also established for yam (Dioscorea rotundata and D. alata) using nodal explants, but transformation efficiency needs to be improved. A transformation protocol using somatic embryogenic callus for yam is under development.

Development of disease- and pest-resistant transgenic crops
Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in the Great Lakes region of Africa. In the absence of natural host plant resistance, IITA, in partnership with NARO-Uganda and the African Agricultural Technology Foundation, has developed transgenic banana by constitutively expressing the Hypersensitive Response Assisting Protein (Hrap) or plant ferredoxin-like protein (Pflp) gene from sweet pepper (Capsicum annuum). The transgenic plants have exhibited strong resistance to BXW in the laboratory and screenhouse tests. The best 65 resistant lines were planted in a confined field trial at the National Agricultural Research Laboratories (NARL), Kawanda, Uganda, for further evaluation.

Transgenic technologies provide a platform for controlling diseases in banana, cassava, and cowpea. Photo by IITA.
Transgenic technologies provide a platform for controlling diseases in banana, cassava, and cowpea. Photo by IITA.
Based on results from mother plants and their first ratoon plants, 12 lines were identified that show absolute resistance. The plant phenotype and the bunch weight and size of transgenic lines are similar to those of nontransgenic plants. These lines will be further tested in a multilocation trial in Uganda. They will be evaluated for environmental and food safety in compliance with Uganda’s biosafety regulations, risk assessment and management, and procedures for seed registration and release, and are expected to be released to farmers in 2017.

Cassava brown streak disease (CBSD) has emerged as the biggest threat to cassava cultivation in East Africa. As known sources of resistance are difficult to introgress by conventional methods into the cultivars that farmers prefer, the integration of resistance traits via transgenics holds a significant potential to address CBSD. Of the available transgenic approaches, RNA silencing is a very promising strategy that has been successfully employed to control viral diseases. IITA, in collaboration with Donald Danforth Plant Science Centre (DDPSC), USA, is developing CBSD-resistant cassava for East Africa.

Nematodes pose severe production constraints, with losses estimated at about 20% worldwide. Locally, however, losses of 40% or more occur frequently, particularly in areas prone to tropical storms that topple the banana plants. IITA, in collaboration with the University of Leeds, UK, has generated transgenic plantain using maize cystatin that limits the digestion of dietary protein by nematodes, synthetic peptide that disrupts chemoreception, or both of these traits. These lines expressing the transgenes were challenged in a replicated screenhouse trial with a mixed population of the banana nematodes, Radopholus similis and Helicotylenchus multicinctus. Many lines were significantly resistant to nematodes compared with nontransgenic controls. The promising transgenic lines showing high resistance will be planted in confined fields in Uganda for further evaluation in mid-2012.

Transgenic technologies for abiotic stress tolerance
Cassava roots undergo rapid deterioration within 24–48 hours after harvest, the so-called postharvest physiological deterioration (PPD), which renders the roots unpalatable and unmarketable. IITA, in collaboration with the Swiss Federal Institute of Technology (ETH) Zurich, is developing cassava tolerant of PPD through the modification of ROS (reactive oxygen species) scavenging systems. The potential is being assessed of various ROS production and scavenging enzymes, such as superoxide dismutase, dehydroascorbate reductase, nucleoside diphosphate kinase 2, and abscisic acid responsive element-binding protein 9 genes, to reduce the oxidative stress and the extent of PPD in transgenic cassava plants.

Future road map
Efforts at IITA over the last 10 years to establish transformation protocols for all the IITA crops have been paying off and have led to the establishment of a genetic transformation platform for cassava, banana/plantain, and yam―the three most important food crops in sub-Saharan Africa. These technologies have contributed to significant advances in incorporating resistance to pests and diseases in banana and cassava. Some of these technologies have the potential to offer additional benefits. For instance, the transgenic technology to control Xanthomonas wilt may also provide an effective control of other bacterial diseases of banana (Moko, blood, and bugtok diseases), and of bacterial blight in other crops such as cassava and cowpea.

Jacob Hodeba Mignouna: Leading the way in science

Mignouna
Jacob Hodeba Mignouna

Jacob Mignouna is a molecular biologist/biotechnologist with an MSc degree in chemical engineering and a PhD in molecular biology and genetics, both from the Catholic University of Louvain, Belgium.

He joined IITA in 1992, as a research scientist-biotechnologist. He led the Biotechnology Unit and developed and implemented a research program on the use of molecular genetic tools to improve food crops and efficiently manage crop genetic resources.

He was a distinguished Frosty Hill Research fellow and had worked as visiting scientist at the Institute for Genomic Diversity, Cornell University, Ithaca, New York; Research Associate Professor of Biotechnology and co-Director of USAID’s Farmer-to-Farmer program in East Africa at Virginia State University, Petersburg, Virginia; and Biosafety Consultant for the USAID Program for Biosafety Systems (PBS), International Food Policy Research Institute (IFPRI), Washington D.C., USA.

As Technical Operations Manager at African Agriculture Technology Foundation (AATF), he identifies opportunities for agricultural technology interventions, assesses the feasibility and probability of success of project concepts, identifies sources of appropriate technologies, negotiates their access and deployment, and provides overall leadership in the implementation of AATF’s project portfolio.

farmers-meetingPlease describe AATF’s work and your work.
AATF is a not-for-profit organization that facilitates and promotes public-private partnerships for the access and delivery of appropriate proprietary agricultural technologies for use by resource-poor smallholder farmers in sub-Saharan Africa (SSA).

The Foundation is a one-stop-shop that provides expertise and know-how that facilitates the identification, access, development, delivery, and use of proprietary agricultural technologies.

AATF works toward food security and poverty reduction in SSA, and its structure and operations draw upon the best practices and resources of both the public and private sectors.

It also contributes to capacity building in Africa by engaging African institutions to work in partnership with others.

AATF strives to achieve sustainable impact at the farm level through innovative partnerships that bring together players all along the food value chain—from smallholder farmers to national agricultural systems, regional and international research organizations, and technology developers.

Currently, AATF is working on biotechnology projects focusing on maize, cowpea, banana, rice, and sorghum—all important crops in Africa. We are also looking at ways to address aflatoxin contamination in peanuts and cereals and processing of cassava.

How did the AATF and IITA partnership come about?
In 2004, IITA approached AATF seeking to access candidate genes conferring resistance against banana bacterial wilt (BXW). IITA had already established contact with Academia Sinica, Taiwan, which held patents to the technology and wanted AATF to negotiate for a license to the ferrodoxin-like protein (pflp) and hypersensitive response assisting protein (hrap) genes from the institute.

In August 2005, IITA, Uganda’s National Agricultural Research Organisation (NARO), and AATF convened a two-day consultative meeting at which stakeholders, including other national research institutes from the Great Lakes region, including IRAZ and other NARS in the region, drafted a project concept note on developing banana bacterial wilt-resistant germplasm.

Soon after, AATF approached Academia Sinica, Taiwan, to license the pflp and hrap genes to it on humanitarian basis.

The initiative has since grown into a full-fledged project designed to enable smallholder farmers in Africa have access to disease-resistant high-yielding banana developed from East African highland varieties.

The project has two components. One focuses on developing transgenic varieties using the acquired technology and the other on improving the capacity of institutions in the region to produce high-quality disease-free planting materials using tissue culture technique.

AATF coordinates the project, including providing support in management of intellectual property rights and regulatory issues, while IITA leads the research, working with Academia Sinica and various institutions, including NARO-Uganda and IRAZ (the national research institution of Burundi), and public and private tissue culture laboratories in Kenya, Tanzania, Uganda, Burundi, Rwanda, and DR Congo.

Through the collaborative research, five banana cultivars—Kayinja, Nakitembe, Mpologoma, Sukali Ndizi, and Nakinyika—have been transformed using an Agrobacterium-mediated system. Several transgenic lines have been produced and tested in vitro by artificial inoculation with the pure Xanthomonas campestris pv. musacearum (Xcm) bacterial culture. Some of the promising lines showed no bacterial wilt symptoms. These plants were further analyzed and confirmed to have the transgene pflp integrated into the banana genome.

With progress on banana transformation well under way, AATF will soon commission a biosafety study. The findings will inform stakeholders as they develop a roadmap for the various processes required for regulatory approvals as the project progresses through the product development pipeline.

Farmers preparing cassava leaves for silage. Photo by S. Kolijn
Farmers preparing cassava leaves for silage. Photo by S. Kolijn, IITA

Please share your insights on collaboration and partnership.
First, collaboration works well if there is a clearly articulated and shared need for joint effort.

Secondly, such partnerships work best if roles and responsibilities are well defined. Work in the banana project is governed by a Memorandum of Association that recognizes the capacities of the partner organizations and facilitates each to contribute optimally to the project.

Also important is the need to bring on board potential partners early enough so that they can provide their input into the project design right from the concept stage. In this project, and generally in all AATF initiatives, we have found comprehensive consultations with a wide range of stakeholders, especially at the formative stage to be a critical success factor.

Third is information flow. Building a communication strategy into the project design ensures that the information needs of partners and external stakeholders are adequately met.

Capacity building is core to all AATF partnerships because of the key role it plays in moving the technologies through the entire food value chain, including scaling up of technologies. In this project, the hub of banana transformation work is at Kawanda, where IITA researchers are working with scientists from national research systems and jointly carrying out the transformation work. This kind of collaboration ensures that staff of national agricultural research institutes in the target countries provide continuity of work in their home country.

Another important aspect of partnership is focus on the smallholder farmer. We have found that having a shared commitment to improve the livelihoods of resource-poor farmers—a clear statement about the ultimate focus of our work—enhances stakeholders’ commitment to project activities.

Then, of course there is the need to have clear negotiated ways to deal with conflict, ensure accountability, and other governance issues.

How did AATF handle the licensing agreement for using the genes for developing resistance to Xanthomonas wilt in bananas?
AATF typically follows a strategy in which it takes the role of the principal and “responsible party” in facilitating public-private partnerships. AATF has entered into licensing agreements to access and hold proprietary technologies and to ensure freedom to operate (FTO) for all the components of the technologies. The Foundation then sublicenses partner institutions to carry out research and adapt technologies for regulatory compliance, and to produce and distribute the technologies. After signing the relevant agreements allowing use of the technology, AATF and partners are guided by a business plan that spells out the roles of each partner and how the technology will be used.

As the principal party, AATF monitors compliance with the requirements of sublicenses to minimize the risk of technology failure, and facilitates the work of appropriate partner institutions to ensure that links in the value chain are connected and result in technology products that reach smallholder farmers.

How would this research impact on banana producers and consumers in Africa?
Millions of people across the East African highlands depend on banana for their livelihoods, directly for food and smallholder producers for the market or as traders and other players in the crop’s value chain. Since banana Xanthomonas wilt broke out in the region, it has caused losses estimated at over US$500 million in Uganda, eastern DR Congo, Rwanda, Kenya, and Tanzania.

In parts of Uganda, where the crop is a staple, some families reported that their banana production had decreased by up to 80%. Given the severity of losses caused by BXW and the fact that the effectiveness of existing remedies is limited, development of disease-resistant varieties will have a huge impact on livelihoods. The benefits can be multiplied many times over by making available clean planting materials to enable farmers to rapidly expand their production.

Increased production will lead to higher incomes for families from sale of the crop, including to the vastly untapped European and American markets, now dominated by South American countries, which account for 60% of the global banana trade.

Scientists inteviewing cassava and maize farmers. Photo by K. Lopez
Scientists inteviewing cassava and maize farmers. Photo by K. Lopez, IITA

What are some of the biggest constraints to adoption of biotechnological tools or products in Africa?
I believe that properly applied agricultural biotechnology holds the key to food security in Africa. Molecular genetics tools should be used not only to improve crops but also to create a better understanding of the abundant diversity of African genetic resources for food, feed, medicine, etc. The biggest constraints to adoption of biotech tools include limited resources—both infrastructural and in terms of trained scientists and other personnel.

Some African countries also lack a regulatory environment conducive to biotech research and development. Although there have been positive changes over the past couple of years, a lot more needs to be done in these areas, including developing regulations to operationalize biosafety laws.

What could be done to take advantage of opportunities that current agricultural technologies provide and harness them for the development of African agriculture or the improvement of food security in SSA?
There are various ways but a key one is by building partnerships, such as those AATF facilitates, that can help access needed technologies, move them from product development and into the hands of farmers. This means different organizations working together to identify and resolve farmer constraints through pooling of available resources where necessary.

We also need to rapidly enhance our capacity to use biotech research. African governments and institutions need to come together and harness their various strengths to develop biotech infrastructure on the continent.

This means training more high-level scientists, equipping laboratories that can serve as centers of excellence and strengthening collaboration among African institutions and between them and research centers and universities abroad.

Lack of awareness about biotech is a major challenge. There is a need for well-designed communication campaigns not only to increase awareness and knowledge of biotechnology, but to increase public acceptance and use of technologies.

You used to head the Biotech Unit at IITA. Please tell R4D Review about your experiences in using biotechnology tools then.
The focus of the Biotechnology Unit, which comprised seven scientists and 45 support staff, was to use the tools that were then available for improving IITA mandate crops. Our work was mainly in two areas. One was developing genetic markers for the characterization of genetic resources, molecular breeding for pests and disease characterization, and exchange of germplasm. The other area was genetic engineering, where we applied tools to address intractable pests and diseases, such as insects that affect cowpea, viral and fungal diseases affecting plantain and banana and cassava mosaic disease. We also addressed diseases and pests in yam, another important food.

What are your aspirations for Africa?
My vision is to see Africa embrace all available tools, including biotechnology and develop the capacity to use them to produce enough food and improve the livelihoods of communities across Africa.

Contact: h.mignouna@aatf-africa.org

Biotech in Nigeria: The journey so far

IITA scientist in Biotech Lab. Photo by O. Adebayo
IITA scientist in Biotech Lab. Photo by O. Adebayo
Nigeria, the world’s largest grower of cassava, producing over 40 million tons per year, is seeking to adopt the use of modern biotechnology tools in agriculture, but efforts are stymied by the absence of a biosafety law.

The passage of the bill by the Nigerian Parliament will launch the country into the production and commercialization of genetically modified organisms (GMO) with the capacity to increase crop production, ensure food security, and improve rural livelihoods.

“The passage of the bill will be great,” said Dr Oyekanmi Nash, Program Director, West African Biotechnology Workshop Series. “Biotechnology holds the key to some of our problems in agriculture and health, and the earlier we tap into it, the better,” he added.

Background
Currently, Nigeria’s population of more than 140 million with an annual growth rate of 2.9% demands increased agricultural production to guarantee food security.

This means traditional agricultural practices, characterized by the use of poor seedlings, must give way to modern tools to allow agriculture to grow by double digits from the current average of about 6%. Such a growth will conserve government revenues from being used in importing food items.

According to government figures, the country spends about US$3 billion annually on food importation. The situation was worse in 2008 when food prices hit the roof, aggravated by the negative effects of severe drought on agricultural production in the northern parts of the country, and high energy costs when crude oil reached $150 per barrel.

“With the turn of events now and for us to meet our food demand in the future, we should apply modern biotech in crop production,” Nash said.

He commended IITA for setting up a modern biotech laboratory in Nigeria, saying that the establishment of such a multimillion dollar laboratory in Nigeria was a reflection of the institute’s commitment to fight poverty in Africa and improving rural livelihoods.

Fluorescence-based genotyping for DNA fingerprinting of plants and pathogens. Photo by IITA
Fluorescence-based genotyping for DNA fingerprinting of plants and pathogens. Photo by IITA
Challenges in introducing GMOs
If the biosafety bill is passed, Nigeria will join other African nations, such as Burkina Faso, Egypt, and South Africa in cultivating GMO crops.

It is expected that the entrance of GMOs will increase crop productivity, lower the cost of production, guarantee food security, and improve both the health and livelihoods of resource-poor farmers who make up more than 70% of the rural population.

The absence of a biosafety law is the problem. In addition, research and development in GM crops are indeed in their infancy in Nigeria as very few establishments in the national agricultural research system have developed the critical mass of human capacity and the infrastructural requirements that would lead to the accelerated development of transgenic materials.

A communiqué issued last year by stakeholders, including the National Biotechnology Development Agency (NABDA) said that other limitations in the commercialization of GMO crops included poor capital equipment, irregular energy, inadequate water supply, and ineffective use of information and communication technology, among others.

The meeting further noted the obvious deficiencies in both the teaching and learning curricula at all school levels and accordingly recommended vibrant and dynamic curricula to generate appropriate labor to meet research and development needs in biotechnology activities.

biotech-milestonesBiotech and Nigeria’s vision 2020
In the next 11 years, Nigeria intends to be ranked among the top 20 economies of the world. Achieving this goal requires adopting policies and options that will lead to improved agriculture and food security among other benefits.

For Nigeria, experts say this will include genetic improvement in the priority crops such as sorghum, cassava, cotton, yam, banana, plantain, maize, wheat, gum arabic, cowpea, and soybean that are of critical importance to the nation.

Prof. Bamidele Solomon, Director-General of NABDA, which has the mandate to promote, coordinate, and regulate biotechnology across the country, said his agency would ensure that the cutting-edge technology of biotech promotes a healthy environment, ensuring national food security and providing affordable health care delivery as well as the alleviation of poverty.

While 2020 appears rather far away, not taking proactive steps toward tackling the present challenges facing the full implementation of biotech will certainly make Vision 2020 a mirage, as far as food security is concerned. This is indeed a wake-up call. The earlier we act, the better.

Is biotechnology a panacea?

cover_photo1Biotechnology is often understood to mean a single technology. In reality it is a collection of technologies that can be applied to address many challenges in agriculture (crop and animal health, food production) pharmaceuticals, and medicine. Biotechnology is often seen as a panacea which is not the case; it is one more tool, albeit an important one, in the arsenal of tools used against the challenges humanity faces. In agriculture, the technology can help accelerate the development of crops resistant to insects and disease, the development of new uses for agricultural products, livestock vaccines, and improved food qualities. African institutions from Cairo to Cape Town, from Dakar to Dar-es-Salaam are using biotechnology in diverse ways.

IITA’s position on biotechnologies is similar to that on all other sciences. We think Africa, its ministries, universities, teaching hospitals, and other research institutions, should not be excluded from any science. Just the need to know, so as to advice governments on the usefulness of a technology to a country’s needs, requires their involvement and knowledge in that science. Whether a particular product of that technology, e.g., genetically modified crops, is adopted or not, is a decision made by governments and not by scientists.

A vibrant local market in Ibadan, Nigeria. Photo by IITA
A vibrant local market in Ibadan, Nigeria. Photo by IITA

Although many African governments are on the brink of embracing the promised benefits of biotechnology, they have not totally committed in terms of providing government funding for more research in agricultural and social/economic development, or policy support for science. What is needed is for R4D institutions, such as IITA and its partners to continue to provide knowledge about these important technologies and their possible impact on sub-Saharan Africa.

This issue highlights some of the cutting-edge work that IITA and its partners (AATF, NARS, donors, NGOs) are doing to help find solutions to problems in tropical agriculture, and thus provide more food and improved livelihoods for the millions of people depending on agriculture. The R4D Review welcomes feedback and comment about any of the information and work featured in this issue. We encourage you to visit the online R4D Review at www.r4dreview.org.

“IITA does not and has not approved or disapproved the use of GM crops in any country. IITA uses all available scientific tools and approaches in its attempt to address hunger and poverty, but the decision to reject or approve and adopt any GM products is the domain and responsibility of the respective national governments. IITA, and rightly so, has no say in such a decison. Any comments to the contrary misrepresent the facts.” —Hartmann, IITA Director General [updated from print version on 25/03/09 ED]

Leena Tripathi: Looking after the welfare of smallholder banana growers

Leena Tripathi was born and grew up in India. She gained a PhD in Plant Molecular Biology from the National Botanical Institute, Lucknow, after completing an MSc in Molecular Biology and Biotechnology at G.B. Pant University of Agriculture and Technology, Pantnagar, India.

She joined IITA in 2000 and worked first in Nigeria and currently in Uganda where her primary research focuses on the development of transgenic Musa spp. with disease and pest resistance. She has established strong links with national and regional partners, and advanced labs. She is also Guest Faculty at the United Nations Industrial Development Organization (UNIDO) for biosafety courses.

Please describe your research work.
Since 2000, I have been developing transgenic banana and plantain resistant to pests and diseases. Currently, I am leading projects on producing bananas resistant to Xanthomonas wilt using the transgenic approach. I am also involved in capacity building in biotechnology and biosafety. I have trained several African scientists in genetic transformation and tissue culture. I have assisted in building capacity on genetically modified organism (GMO) detection and biosafety in East Africa by training students and national scientists on banana transformation and molecular biology. And I would like to acknowledge the strong financial support from donors such as Gatsby Charitable Foundation, African Agricultural Technology Foundation (AATF), US Agency for International Development, and the UK Department for International Development (DFID); and IITA of course.

Why did you choose to work in Africa?
Africa has missed the Green Revolution but should not miss the Gene Revolution. For this it needs human capacity in biotechnology that will help to accomplish things that conventional plant breeding could never do. The public needs to be better informed about the importance of biotechnology in food production.

What is the importance of transgenic technologies in banana improvement?
Many pests and diseases significantly affect banana cultivation and cause crop losses worldwide. Development of disease-resistant banana by conventional breeding remains difficult for various technical reasons. Transgenic technologies are the most cost-effective approach. There are enormous potentials for genetic manipulation using appropriate transgenes from other plants to achieve objectives in a far shorter time. It may also be possible to incorporate other characteristics such as drought tolerance, thus extending the geographical spread of production.

How do you demystify or explain a concept like biotechnology to lay audiences?
People think that biotechnology is just genetic modification (GM) technology. Contrary to its name, biotechnology is not a single technology; it is a group of technologies that uses biological systems, living organisms, or their derivatives, to make or modify products or processes for specific use. This includes recombinant DNA technology, genetic engineering, GM foods, biopharmaceuticals, bioremediation, and more.

Biotechnology is not new; it has flourished since prehistoric times. When the first human beings realized that they could plant their own crops and breed their own animals, they learned to use biotechnology. The discovery that fruit juices fermented into wine, or that milk could be converted into cheese or yogurt, or that beer could be made by fermenting solutions of malt and hops, started the study of biotechnology. When the first bakers found that they could make soft, spongy bread rather than a firm, thin cracker, they were acting as fledgling biotechnologists.

“Modern” biotechnology derives from techniques discovered only in the last 20 years. These include the ability to cut and stitch DNA, to move DNA and genes from one organism to another, and to persuade the new gene in this new organism, that is to make new proteins. Genetic engineering technology is a branch of modern biotechnology and involves the transfer of gene(s) from one organism to another to create a new species of crops, animals, or microorganism. Modern biotechnology has offered opportunities to produce more nutritious and better tasting foods, higher crop yields, and plants that are naturally protected from disease and insects.

What have you learned on the job?
I joined IITA as a biotechnologist with plenty of experience in research but not in the field. Working at IITA has been overwhelmingly positive. I have gained experience in both research and administration. I have learned to appreciate the benefits of working in multidisciplinary and multicultural teams and of linking research to farmers in the field. I can now write successful project proposals, get funding, lead projects, and disseminate results to national partners and finally to farmers. Good communication skills are essential for successful research. One needs to be a good team worker and establish strong and successful partnerships as we are doing at IITA-Uganda. When I was relocated here, I realized the situation was very different. IITA in Ibadan has facilities but in Uganda, IITA facilities are based within a national partner, the National Agricultural Research Organization. I wanted to learn quickly from the experiences of others so I talked to colleagues about their work and successes and to national scientists about their expectations. I learned quickly.

Any advice for IITA colleagues?
IITA scientists should be committed to provide strong leadership in the key research areas to ensure scientific excellence and the quality of products. They should work applying “new science” to enhance food security and income generation for resource-poor farmers.

What are your future research plans?
I want to evaluate the disease resistance of banana varieties in the field, evaluate transgenic plants in the confined field for efficacy against Xanthomonas wilt disease, with the University of Leeds develop nematode-resistant plantains, and develop varieties with multiple disease resistance by integrating several genes with different targets or modes of action into the plant genome. I also want to train more national staff/students to build capacity in the region.

What is your formula for success?
The addition and sometimes multiplication of five key elements: vision, strategy, confidence, hard work, and learning. I am focused and have a clear vision for my research, based on project outputs. I frame strategy with clear goals. I follow the strategy with my group members and work hard to achieve the goals. At each step I identify problems and learn to solve or avoid them so that the group moves smoothly and fast to achieve the goals. I set the goals for my group at the start of each year. Everyone works extra hours to achieve group goals. I do not hesitate to seek advice and suggestions from experts, superiors, and collaborators to move things efficiently. Support is very important. I have benefited from support and encouragement from my superiors, higher IITA management, donors, collaborators, and from family. IITA nominated me for the CGIAR Young Scientist award in 2005 and gave me their Top Scientist award, based on my research achievements.