Embryo rescue and anther culture for breeding new yam varieties

Yukiko Kashihara, y.kashihara@cgiar.org

To contribute to reducing poverty and increasing food security, IITA is breeding new varieties of yam based on demand and value addition. This is usually done by crossing two parents of the same species (intraspecific breeding, e.g., Dioscorea rotundata clones, TDr Ehuru × TDr Ehobia) or different species (interspecific hybridization, e.g., D. rotundata clone, TDr Ufenyi × D. alata clone, TDa 85/00250) to transfer useful traits. However, intraspecific or interspecific yam breeding is still being constrained by poor flowering, low seed-setting, a low rate of seed germination, and differences in flowering periods as male and female parents flower at different times.

Interspecific hybridization is particularly useful to produce additional variations in Dioscorea species. For instance, interspecific hybridization of D. alata and D. rotundata can contribute to the transfer from D. rotundata to D. alata of genes conferring tolerance to anthracnose disease. However, interspecific hybridization is still a challenge. One example in Japan dealt with the hybridization between D. japonica and D. opposita produced with the aid of the embryo rescue technique (Araki et al. 1983). In addition to interspecific hybridization, the use of homozygous parents (having the same pairs of genes) could add efficiency to breeding. Anther culture can also produce homozygous plants through chromosome doubling of haploid plants. Establishing an anther culture system will further diversify breeding approaches. Therefore, current studies are focusing on (i) refining the embryo rescue technique to improve the efficiency of interspecific hybridization, and (ii) establishing a method for producing haploid yam which saves several generations in the breeding program.

Using ovule culture (excised ovules from the ovary and cultured on media), we obtained one plant which resulted from a cross in 2012. However, after flow cytometric analysis, we found that the plant was derived from an ovule parent. For anther culture, more investigation is needed to find out the optimum medium and conditions. The establishment of reliable tissue culture protocols and efficient working schemes is essential. The success of this will contribute to allowing wide hybridization and saving time and space for IITA’s yam improvement program. Moreover, having haploids will be advantageous for yam research, especially in gene mapping, genomics, and other applications.

Reference

Araki, H. et al. 1983. Some Characteristics of Interspecific Hybrids between Dioscorea japonica Thunb. and Dioscorea opposite Thunb. Japanese Society for Horticultural Science 52:153-158.

Genetic transformation of yam

Leena Tripathi, l.tripathi@cgiar.org

Yam (Dioscorea spp.) is a multi-species, polyploid and vegetatively propagated tuber crop in the tropics and subtropics that provides food security and income to over 300 million people. There are 600 Dioscorea species; however, only a few of them are regularly cultivated for food. Dioscorea rotundata and D. cayenensis (both known as Guinea yam) are most popular and economically important in West and Central Africa where they are indigenous, while, D. alata (known as water yam) is the most widely distributed species globally. Yam is the second most important root and tuber crop in sub-Saharan Africa after cassava in terms of production with about 57 million metric tons. Over 95% of world yam production occurs in the yam belt of West and Central Africa with Nigeria alone accounting for about 66% of the world’s total. Some wild yam species are also known to produce secondary metabolites of pharmaceutical importance such as steroidal sapogenin, diterpenes, and alkaloids.

Despite the crop’s economic and sociocultural importance, its cultivation is generally limited by high costs of planting material and labor, decreasing soil fertility, low yield potential of varieties, and increasing levels of field and storage pests (nematodes) and diseases (anthracnose, tuber rots, and yam virus complex). In West Africa, about 11 million tons of yam are lost annually because of damage in storage initiated by nematodes. Plant-parasitic nematode damage is a critical factor in tuber quality reduction and yield loss in yam, both in the field and in storage, which is perpetuated over seasons through infected seed material. Nematodes also facilitate fungal and bacterial attacks.

Nematodes can be managed by nematicides, but are not commonly used due to their high cost. As yam is vegetatively propagated, nematode-affected tubers are transferred in infected seed yam material. Yam nematodes reproduce and build up large populations in stored tubers, causing severe damage and facilitating fungal and bacterial attacks that cause anthracnose disease, dry rot, soft rot, and wet rot. It is therefore necessary to control plant-parasitic nematodes to increase or at least maintain reasonable yields of yam and protect susceptible germplasm from total loss. Nematode-resistant varieties of yam can be very effective for nematode control.

Genetic transformation is an alternative tool for developing nematode-resistant varieties. This option is also important because host plant resistance has not yet been found in the major cultivated species (D. rotundata, D. cayenensis, D. alata) or close relatives with which they can be crossed by conventional breeding and selection for the trait. It is therefore necessary to control plant-parasitic nematodes to increase or at least maintain reasonable yields of yam and preserve susceptible germplasm.

Genes conferring nematode resistance are already available from the University of Leeds for plantain (Musa spp.) transformation at IITA, which can easily be made available and assessed against nematodes in yam. This can only be possible after the yam transformation system is established. To date, only a single report has been published on the stable transformation of D. alata by particle gun using reporter gene (Tor et al. 1993), and this still needs improvement. Tor et al. (1998) also reported transient gene expression in protoplast of Dioscorea spp. using polyethylene glycol (PEG)-mediated direct uptake method. The stable transformation of yam using PEG-mediated uptake still needs to be developed.

There is no report on Agrobacterium-mediated transformation of D. alata and D. rotundata, which is the preferred method for genetic engineering of plants, offering several advantages over direct gene transfer methodologies (particle bombardment, electroporation), such as the possibility of transferring only one or a few copies of DNA fragments carrying the genes of interest at higher efficiencies with lower cost and the transfer of very large DNA fragments with minimal rearrangement.

The development of stable transgenic plants requires an efficient regeneration system amenable to genetic transformation and stability of transgene under field conditions. Regeneration systems from meristems of D. rotundata and D. alata have recently been established at IITA (Adeniyi et al. 2008; Tripathi et al. unpublished). Recently, direct shoot organogenesis was also reported in petiole explants of D. rotundata, D. cayenensis, and D. alata (Anike et al. 2012). These regeneration systems are yet to be evaluated for their amenability to transformation. Regeneration through callus or somatic embryogenesis, which is ideal for transformation, remains to be established. There are only a few reports on plant regeneration from embryogenic cell cultures of Chinese yam (D. opposita), D. alata, and D. cayenensis (Belarmino and Gonzales 2008; Nagasawa and Finer 1988; Twyford and Mantell 1996; Viana and Mantell 1989). However, there is no report of regeneration from somatic embryogenesis of D. rotundata. We have obtained embryogenic callus but further research is needed to develop an efficient regeneration protocol using callus.

As a transformation system for yam is currently not available, therefore, IITA, with support from the Bill & Melinda Gates Foundation, is conducting research to develop a regeneration and transformation system for yam varieties most preferred by farmers. Once a transformation system for yam is established, the protocol will then be used for producing nematode-resistant varieties for effective control of this major pest in yam production systems.

References

Adegbite AA et al. 2006. Survey of plant-parasitic nematodes associated with yams in Edo, Ekiti and Oyo states of Nigeria. African J Agric Res 1: 125-130.

Adeniyi OJ et al. 2008. Shoot and plantlet regeneration from meristems of Dioscorea rotundata poir and Dioscorea alata L. African J Biotechnol 7: 1003-1008.

Anike FN et al. 2012. Efficient shoot organogenesis in petioles of yam (Dioscorea spp.). Plant Cell Tiss Organ Cult 111:303–313.

Belarmino MM et al. 2008. Somatic embryogenesis and plant regeneration in purple food yam (Dioscorea alata L.). Ann Trop Res 30:22-33.

Bridge J et al. 2005 Nematode parasites of tropical root and tuber crops. In: Luc, M., Sikora, R.A. and Bridge, J. (eds) Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, 2nd edn. CABI Publishing, Wallingford, UK, pp. 221-258.

Nagasawa A, Finer JJ. 1988. Plant regeneration from embryogenic suspension cultures of Chinese yam (Dioscorea opposite thumb.). Plant Sci 60:263–271.

Tor M et al. 1993. Stable transformation of the food yam Dioscorea alata L. by particle bombardment. Plant Cell Rep 12: 468-473.

Tor M et al. 1998. Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies. Plant Cell Tiss Organ Cult 53:113–125.

Twyford CT, Mantell SH. 1996. Production of somatic embryos and plantlets from root cells of greater yam. Plant Cell Tissue Organ Cult 46:17–26.

Viana AM, Mantell SH. 1989. Callus induction and plant regeneration from excised zygotic embryo of the seed propagated yams Dioscorea composite Hemsl. and D. cayenensis Lam. Plant Cell Tissue Organ Cult 16: 113–122.

Novel approaches for the improvement of yam germplasm conservation and breeding

Gezahegn Girma, g.tessema@cgiar.org

IITA, Ibadan, Nigeria

Genetics and Biotechnology Lab, Plant and AgriBiosciences Research Centre (PABC), School of Natural Sciences, National University of Ireland Galway, Ireland

Efforts are ongoing at IITA, in collaboration with the National University of Ireland Galway, to understand the genetic diversity, evolutionary relationship of yam species, flowering and sex-related genes, polyploidy and its effect on phenotypic performance and somaclonal variation in in vitro regenerated plants. This work is expected to contribute to the development of new tools for assessing yam diversity, and understanding genetic and phenotype linkages for improving yam breeding and germplasm conservation. The status of thrust areas being pursued to address these gaps is summarized here.

Identification of chloroplast or nuclear regions that can help discriminate species

Developing molecular tools supported by taxonomic identification is very important for unambiguous species naming or classification. A DNA barcode aids taxonomic identification, which uses a standard short genomic region that is universally present in target lineages and has sufficient sequence variation to discriminate among species. The single locus rbcL, matK, combination of rbcL+ matK, the noncoding intergenic spacer, trnH-psbA of chloroplast regions, and ITS of nuclear region were evaluated using a criterion for candidate DNA barcode for Dioscorea species identification. All the sequences were assessed for universality (ease of PCR amplification and sequencing), sequence quality, and species discriminatory power.

Genetic polymorphism of cultivated guinea yams and their relationship with wild relatives

Next generation-based genotyping procedures such as genotyping by sequencing (GBS) is now considered as an excellent tool in plant genetics and breeding (Poland and Rife 2012) due to its genome-wide molecular marker discovery, genotyping for multiplexed samples, flexibility, and low cost. A study was conducted to understand the genetic diversity within and between two cultivated guinea yams and five of its wild relatives (D. praehensilis, D. mangenotiana, D. abyssinica, D. togoensis, and D. burkilliana) using GBS, morphology, and ploidy analysis.

In general, the genetic contribution of wild relatives, origin of cultivated species, ongoing domestication practices, potential polyplodization process and utility of GBS in generating genotypic information were demonstrated. Similarly, GBS could further be used for understanding of genetic relationship studies of other species within the genus Dioscorea that holds a large number of species in addition to the guinea yams. Due to the cost effectiveness of GBS, there is major potential for the yam genebank collection in IITA (and other yam germplasm collections) to be genotyped using the GBS procedure. GBS can help to indicate the level of genetic diversity and guide the need for more germplasm collection, duplication, and mismatch identification.

Understanding the molecular genetics of flowering

D. rotundata are mostly dioecious, with separate male and female plants, although a few lines are identified as monoecious. It is also common to find nonflowering, which is perhaps dominant. The dioecy of the crop makes the synchronization of flowering time very difficult. For the efficient improvement of yam crops through conventional breeding, understanding better the genetic mechanisms of flowering in Dioscorea is essential. The tiny and large numbers of Dioscorea chromosomes is also a challenge to make critical observations. Therefore identification of the sex-determining chromosomes is difficult at cytological level. In addition, the genes that control the flowering in yam are not yet known. A study is being conducted to identify gene expression patterns in relation to different flowering habits (male, female, and monoecious) of D. rotundata accessions using the SuperSAGE technique. The study outcome is expected to help understand the flowering biology of the crop in general and once confirmed, identified sex-determining candidate genes can be incorporated in varieties with inconsistent nonflowering to produce regularly flowering cultivars, and hence, improve yam production.

Morphological, ploidy and molecular diversity

The section Enantiophyllum of the genus Dioscorea is generally known to produce only 1-3 underground tubers. Hence, there is a need for exploitation of other options of planting materials such as aerial tubers as an alternative planting material to underground tubers. The study was conducted to investigate the molecular, morphological, and ploidy variation across Dioscorea alata accessions producing aerial tubers in comparison with accessions without aerial tubers. The aerial tuber production of accessions was found correlated with ploidy level, distinct morphological characteristics, and SSR analysis discriminated according to its pattern of aerial tuber production.

Evaluating somaclonal variation under in vitro regeneration

In spite of the several advantages from the tissue culture system in plant ex situ conservation, somaclonal variation is regarded as one of the major problems of many tissue-cultured plants (Bordallo et al. 2004). On the other hand, somaclonal variation is known for its usefulness in crop improvement by creating novel sources of variability that could result in improved yield, resistance to diseases, and quality improvement. Detection and elimination of undesirable variants and spotting variants with useful agronomic traits is therefore essential. The study on meristem derived in vitro clones of D. rotundata accessions with their original genotypes is ongoing to evaluate and verify somaclonal variation using AFLP markers.

References

Bordallo PN, Silva DH, Maria J, Cruz CD, Fontes EP. 2004. Somaclonal variation on in vitro callus culture potato cultivars. Horticultura Brasileira 22:300-304.

Poland JA, Rife TW. 2012. Genotyping-by-Sequencing for Plant Breeding and Genetics. Plant Gen 5:92-102.

Ecofriendly bioherbicide approach for Striga control

Abuelgasim Elzein, a.elzein@cgiar.org, and Fen Beed

Root parasitic weeds of the genus Striga are a significant constraint to cereal and cowpea production in sub-Saharan Africa. They can cause total crop losses particularly during drought, in infertile soils and cereal monocropping. Striga causes annual losses of US$7 billion and affects incomes, food security, and nourishment of over 100 million people mostly in sub-Saharan Africa.

Each Striga plant can produce thousands of seeds, viable for over 10 years. Their intimate interaction with different host plants prevents the development of a silver bullet control technology that subsistence farmers can adopt. Hence, it is widely accepted that an integrated approach to Striga management is required for which biocontrol represents a crucial component.

Bioherbicide innovation
A bioherbicide is a plant pathogen used as a weed biocontrol agent (BCA), which is applied at sufficient rates to rapidly cause a disease epidemic that kills or severely suppresses the target weed. The use of biocontrol technology to manage Striga is a desirable control method as it is environmentally friendly, safe to farmers and crop consumers, specific to the target host, and has the potential to be economically viable. In addition, biological control also assists in the development of a balance of nature, the creation of more biodiversity, and sustaining of complex ecological interactions.

Since the early 1990s, a series of intensive disease surveys in many countries of sub-Saharan Africa has evaluated hundreds of microorganisms for their pathogenicity and virulence against Striga. Fusarium oxysporum Schlecht isolates have been the most promising. However, the discovery of a highly effective pathogen is only one step in the process of developing bioherbicides, for which the inoculum mass production, formulation, delivery, and storage ability must be optimized, and the mode of action, host specificity, and biosafety evaluated and fully understood.

The most widely studied and used fungal isolate that met all requirements for a potential bioherbicide for Striga is F. oxysporum Schlecht f. sp. strigae Elzein et Thines (isolates Foxy2 and PSM197). These are highly virulent, attack Striga in all growth stages—from seed to germination, from seedling to flowering shoot; protect the current crop yield; and prevent seed formation and dispersal.

F. oxysporum f. sp. strigae is highly host-specific to the genus Striga, and does not produce any known mycotoxic compounds. Thus, its use does not pose health risks to farmers, input suppliers, traders or consumers or threaten crops or the environment. Its unique DNA constitution differs from other forms of F. oxysporum deposited in GenBank, known to cause crop diseases. Indeed, this ensures its biosafety and greatly facilitates its wider application and use as a bioherbicide.

In addition techniques for massive production of inoculum of F. oxysporum f. sp. strigae was optimized based on simple and low-cost methods and using inexpensive agricultural by-products available in sub-Saharan Africa. The chlamydospores produced by this fungus have the advantage of being able to survive extreme environmental events while still remaining viable. This is an important feature required for a BCA suited to hot and dry climatic conditions of cereal production in sub-Saharan Africa, and to produce stable, durable, and pathogenic propagules.

Extensive research by the University of Hohenheim (UH, Germany), IITA (Benin), McGill University (Canada), and Institute for Agricultural Research – Ahmadu Bello University (Nigeria), has enhanced application of F. oxysporum f. sp. strigae, its formulation into bioherbicidal products, and its delivery for practical field application. The Striga bioherbicide contains the Striga host-specific F. oxysporum f. sp. strigae, applied in massive doses to create a high infection and disease level to kill or severely suppress Striga.

Promotion in West Africa
The bioherbicide is a component of the IITA-led project, Achieving sustainable Striga control for poor farmers in Africa, funded by the Bill & Melinda Gates Foundation to intensively promote technologies to combat Striga in sub-Saharan Africa. The project will validate the potential of the bioherbicide seed treatment technology across major Striga-infested agroecological zones and maize-based farming systems, while also confirming the biosafety and developing molecular detection tools. Here are the highlights of the results:

Technology validation: Several multilocation trials were conducted under natural and artificial Striga infestation across two agroecological zones in northern Nigeria to evaluate the efficacy of Striga bioherbicide (F. oxysporum f. sp. strigae). The inoculum produced by UH and SUET seed company was delivered as a film-coat on maize seeds (see below).The application of the bioherbicide technology in combination with Striga resistant maize reduced Striga emergence by 73% and 39%, compared to the susceptible and resistant controls, respectively, and prevented 81% and 58% of emerged Striga plants from reaching flowering and 56% and 42% of the maize plants from attack by Striga (see next page). The combination of bioherbicide with Striga susceptible variety significantly reduced Striga emergence by 53%, resulting in 42% reduction in number of flowering plants and in 21% increase in grain yield compared to the susceptible control.

In addition, disease symptoms were recorded on emerged Striga plants parasitizing maize plants coated by the bioherbicide. The reduction in Striga emergence across maize varieties indicates the effectiveness of the bioherbicide to attack seeds under the soil surface. The synergistic effect of the bioherbicide technology combined with the Striga resistant maize is expected to reduce the Striga seedbank and thus the impact of Striga on subsequent maize crops.

Biosafety: To further ensure the safety of Striga BCA and to demonstrate and increase awareness among farmers, regulatory authorities, and stakeholders, a wide host range study was carried out using 25 crops in collaboration with IAR-ABU and the Nigerian Plant Quarantine Service (NPQS)  under field and screenhouse conditions in Nigeria. Results revealed that none of the test plants showed any infection by the biocontrol agent both in the field and screenhouse, and no detrimental growth effects were measured or visual losses to plant health recorded in any of the inoculated crops tested, i.e., inoculation with the Striga BCA did not cause any delay in emergence, and a decrease in plant height, plant vigor, chlorophyll content per leaf, shoot fresh and dry weight. Hence, the Nigerian regulatory authorities (NPQS, NAFDAC) and other stakeholders were satisfied and confident that no disease was produced on plants other than Striga by the BCAs and that it is safe to use. In addition, a mycotoxin produced by Striga bioherbicide  F. oxysporum f. sp. strigae was analyzed and evaluated by our project partner, the University of Stellenbosch in South Africa. An evaluation of existing isolates of F. oxysporum f. sp. strigae does not produce well-known mycotoxins (e.g., Fumonisin and Moniliformin) that pose a threat to animal or human health. This finding further confirms the safety of this bioherbicide.

Molecular detection tools: Development of a monitoring tool specific to the Striga bioherbicide is important to certify inoculum quality, monitor the presence and persistence of the BCA in soils, and validate its environmental biosafety. UH is developing a monitoring tool.

The AFLP fingerprinting technique was successfully used in developing a primer pair capable of differentiating the F. oxysporum f. sp. strigae group from other Fusarium species. In addition, the monitoring tool has shown a high specificity for isolate Foxy2 and was used to monitor its spread and persistence in rhizobox experiments under different management practices using Kenyan soils. This promising result provides a proper baseline to further the existing primer set.

Bioherbicide + pesticide technology: The novel combination and integration of the bioherbicide technology plus imazapyr herbicide for Striga control with pesticides in a single-dose seed treatment to control fungal pests offers farmers with maize seed that is able to achieve its yield potential. The use of each technology (BCA or imazapyr) has been shown to be effective when applied independently using seed coating techniques, but have not been integrated.

The compatibility of Striga BCAs with different pesticides (herbicides and fungicides with insecticide components) was studied in vitro in the laboratory. Striga BCAs showed excellent compatibility with imazapyr (a herbicide seed coating used in combination with IR maize to control Striga), Metsulfuron Methyl (MSM) (a herbicide seed coating developed by DuPont to control Striga in sorghum), and glyphosate (an intensively used herbicide). A similar result was also achieved with the commonly used seed treatment fungicides at the recommended application doses.

Accordingly, doses and complementary seed coating protocols for the three compatible technologies (BCA, herbicide, and fungicides) have been developed and IR maize seeds were successfully coated with a single-dose seed treatment of BCA inoculums and imazapyr. The results showed that imazapyr did not interfere with the BCA during seed coating, with BCA growth and sporulation after coating, and with IR maize seed germination. Seeds of IR maize varieties can thus be coated with the herbicide and the BCA and then fungicide and delivered to farmers using the same input pathway. Screenhouse and field trials are being carried out to generate data on the combined efficacy of the applied technologies. The demonstrated compatibility of Striga BCA with the different pesticides that contain a wide range of active ingredients indicate that the combination and delivery of the Striga bioherbicide technology with a large number of pesticide products is possible. These findings are expected to provide a triple action seed coating package for direct control of Striga and fungal diseases of maize in sub-Saharan Africa.

Suitability to African farming systems
Our strategy for scaling-up the bioherbicide innovation is based on using technology appropriate to Africa to ensure that sustained production of the bioherbicide is feasible at a cost affordable to African small-scale farmers. The seed-coating treatment requires significantly less inoculums, establishes the BCA in the cereal rhizosphere, i.e., the infection site of Striga, and provides a simple, practical, cost-effective delivery system for adoption by input suppliers to subsistence farmers. Arabic gum as a coating material has been shown to increase the rate of mycelia development and enhance BCA sporulation. Its availability in sub-Saharan Africa at a low price is an additional economic advantage. A commercial seed coating process, developed and optimized at UH with SUET Seed Company in Germany, is being transferred and adapted at IITA, Ibadan, to be used as an experimental production unit for capacity building and as a model for eventual transfer of seed treatment technology to the private sector after validation.

Outlook
One unique advantage of this bioherbicide is that the ability of Striga to become resistant to it is virtually unknown as a consequence of the suite of enzymes and secondary metabolites that the BCA produce to become pathogenic and virulent against the target (Striga). Hence after validation, delivering the bioherbicide technology in combination with resistant maize or with the herbicide imazapyr is expected to increase efficacy in controlling Striga. Bioherbicide and other compatible technologies have different modes and sites of action against Striga, and in a combination they will have a much greater chance of reducing the potential risk of development of resistance to a single technology (resistant varieties or herbicides) used separately and repeatedly.
The potential delivery of coated seeds of resistant maize with bioherbicide in one package to farmers using the same input pathway will reduce transaction and application costs and enhances the economic feasibility and adoptability of the technologies. Similarly, compatibility of BCA with imazapyr and fungicides allow seed coating of IR-maize with bioherbicide, imazapyr, and fungicides with a single-dose seed coating application.

Future plans
Currently, large-scale field testing is ongoing and is being implemented to further validate bioherbicidal efficacy across two agroecological zones where the common scenarios for maize infestation by Striga in northern Nigeria are represented. For understanding of farmers’ preferences and perceptions, socioeconomic analysis and cost-benefit analysis of bioherbicidal technology based on field data/surveys and interviews, current market information, and links with other Striga control strategies will be undertaken. After validation, dissemination and commercialization will be promoted through private sector partnerships and integrated with other control options such as resistant varieties, IR varieties combined with seed treatment with imazapayr, crop rotation with legumes, and soil fertility management practices, to achieve sustainable management of Striga.

Partners
IITA (Dr F. Beed, Dr A. Elzein & Dr A. Menkir), Institute for Agricultural Research – Ahmadu Bello University (Dr A. Zarafi), Nigeria; University of Hohenheim (Prof G. Cadisch, Dr F. Rasche & Prof J. Kroschel), Germany; The Real-IPM Company Ltd (Dr H. Wainwright), Kenya; University of Stellenbosch (Prof A. Vilioen), South Africa; and McGill University (Prof A. Watson), Canada.

References
Beed F.D., S.G. Hallet, J. Venne, and A. Watson. 2007. Biocontrol using Fusarium oxysporum; a Critical Component of Integrated Striga Management. Chapter 21 in Integrating New Technologies for Striga control: Towards ending the Witch-hunt (Ejeta, G. and J. Gressel, eds). World Scientific Publishing Co. Pte. Ltd. pp 283-301.

Ciotola, M., A. DiTommaso, and A. Watson. 2000. Chlamydospore production, inoculation methods and pathogenicity of Fusarium oxysporum M12-4A, a biocontrol for Striga hermonthica. Biocontrol Science and Technology 10: 129-145.

Ejeta, G. 2007. The Striga scourge in Africa: A growing pandemic In: Ejeta, G. and J. Gressel, eds. Integrating New Technology for Striga Control: Towards Ending the Witchhunt. World Scientific Publishing Co. Pte. Ltd., UK. pp. 3-16.

Elzein, A.E.M. 2003. Development of a granular mycoherbicidal formulation of Fusarium oxysporum “Foxy 2” for the biological control of Striga hermonthica. In: “Tropical Agriculture 12– Advances in Crop Research (2)” (J. Kroschel, ed.). Margraf Verlag, Weikersheim, Germany, 190 pp, ISBN 3-8236-1405-3.

Elzein, A., J. Kroschel, and V. Leth. 2006. Seed treatment technology: an attractive delivery system for controlling root parasitic weed Striga with mycoherbicide. Biocontrol Science and Technology, 16(1) 3-26.

Elzein, A., F. Beed, and J. Kroschel. 2012. Mycoherbicide: innovative approach to Striga management. SP-IPM Technical Innovations Brief, No. 16, March 2012.

Kroschel, J. and D. Müller-Stöver. 2004. Biological control of root parasitic weeds with plant pathogens. In: Inderjit, K. (ed.), Weed biology and management. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 423–438.

Kroschel, J., A. Hundt, A.A. Abbasher, J. Sauerborn. 1996. Pathogenicity of fungi collected in northern Ghana to Striga hermonthica. Weed Research 36 (6), 515–520.

Marley, P.S., S.M. Ahmed, J.A.Y. Shebayan, and S.T.O. Lagoke. 1999. Isolation of Fusarium oxysporum with potential for biocontrol of the witchweed Striga hermonthica in the Nigerian Savanna. Biocontrol Science and Technology 9: 159–163.

Venne J., F. Beed, A. Avocanh, and A. Watson. 2009. Integrating Fusarium oxysporum f. sp. strigae into cereal cropping systems in Africa. Pest Management Science 65: 572–580.

CGIAR Research Programs and natural resource management

Bernard Vanlauwe, b.vanlauwe@cgiar.org, Alpha Kamara, Stefan Hauser, and Piet Van Asten

Figure 1. Relationships between Humidtropics and other CRPs.
Figure 1. Relationships between Humidtropics and other CRPs.

Over the past few years, the CGIAR system has been engaged in a substantial, research-led restructuring of its research agenda through the creation of the CGIAR Research Programs (CRP), supported by a Consortium Office, a Fund for international agricultural research, an Independent Science and Partnership Council, and an Independent Evaluation Arrangement. A total of seven CRPs are now active with several having a crop-specific focus, others a farming system focus, and others addressing themes related to natural resource management (NRM) or the creation of an enabling environment for the uptake of improved options. IITA is leading the Humidtropics CRP and contributing significantly to the CRPs on Water, Land, and Ecosystems (WLE) and the Climate Change, Agriculture, and Food Security (CCAFS), all of these having significant NRM components. This article highlights these components in the context of the overall CGIAR research framework and the specific contributions of IITA towards the success of these CRPs.

Humidtropics
The humid tropics is home to 2.9 billion of the world’s poorest people. It is the part of the world with the biggest gap between its ecological and economic potential and human welfare. The Humidtropics CRP aims to realize more of that potential to improve the livelihoods of the majority of the population and protect their environment and natural resources from the usual rapid degradation when used for agriculture or forest (timber) exploitation. Humidtropics seeks intensification pathways and critical points of intervention to design superior crop, livestock, fallow, and perennial (tree) production systems along with improved soil, water, and vegetation management practices, and the identification of investment strategies for sustainable natural resource base management.

Interventions will increase overall farm and system productivity and income while improving the natural resource base, particularly soil quality. Humidtropics will strategically select critical entry points that foster more diverse system components to generate more equitable agricultural growth in which rural communities move beyond commodities, reduce their risks, sustainably manage their natural resources, and effectively reduce rural poverty.

Women farmers are one of the target beneficiaries of the integrated research and development programs that aim to help boost agricultural productivity in the humid tropics. Photo by IITA
Women farmers are one of the target beneficiaries of the integrated research and development programs that aim to help boost agricultural productivity in the humid tropics. Photo by IITA

Humidtropics is led by IITA in partnership with the International Center for Tropical Agriculture (CIAT), International Livestock Research Institute (ILRI), World Agroforestry Centre (ICRAF), International Potato Center (CIP), Bioversity International, International Water Management Institute (IWMI), International Centre of Insect Physiology and Ecology (icipe), Forum for Agricultural Research in Africa (FARA), The World Vegetable Center (AVRDC), and Wageningen University. It will operate in various action areas in Africa, Latin America, and Asia with the Western Humid Lowland and the East and Central African Highland Action Areas led by IITA. Humidtropics is a systems research program that covers all lowland humid and subhumid ecologies (between dry land and aquatic), draws on research in commodity CRPs, and integrates technologies and forecasting ability from the CRPs on Policies and Markets, Nutrition and Health, Water and Land, and Climate Change (see diagram).

Water, Land, and Ecosystems (WLE)
The global population in 2050 will be about 9 billion, with most of the increase between now and then taking place in developing countries. To feed the world in 2050 and beyond, we need to intensify agricultural production. Many observers believe that intensification will cause unacceptable harm to the environment, perhaps undercutting the ecosystems that support agriculture. WLE challenges this perspective and examines how we can intensify agriculture while protecting the environment and lifting millions of farm families out of poverty.

To achieve the vision of sustainable intensification, we must redouble our efforts to increase agricultural productivity, while protecting the environment, and we must conduct new and integrative research on agricultural and ecosystem interactions. Consequently the objective of WLE is to learn how to intensify farming activities, expand agricultural areas and restore degraded lands, while using natural resources wisely and minimizing harmful impacts on supporting ecosystems.

Within the broad topic of WLE, we have identified five strategic research portfolios (SRPs): Irrigated Systems, Rainfed Systems, Resource Reuse and Recovery, River Basins, and Information Systems. The Rainfed Systems SRP, to which IITA is contributing, targets 80% of the world’s farmland that is largely rainfed. Although many farmers in rainfed areas capture and store water for use as supplemental irrigation, millions more entirely depend on rainfall. In many areas, increasing populations have placed substantial pressure on rainfed cropland and on the land and water resources used by livestock. As a result, the land and water resources in many areas are degraded and unproductive. WLE researchers will determine ways to restore degraded resources using multifunctional landscape management approaches, and will develop integrated soil and water management techniques.

In pastoral systems, extensive land degradation and the loss of access to water and land resources threaten the livelihoods of millions of pastoralists, leading to conflicts in some areas. WLE researchers will determine the changes in land and water management and the complementary policies needed to support pastoral livelihoods. The Rainfed System SRP currently works around five problem sets: (1) Recapitalizing African soils and reducing land degradation; (2) Revitalizing productivity on responsive soils; (3) Increasing agricultural production while enhancing biodiversity; (4) Enhancing availability and access to water and land for pastoralists; and (5) Reducing risk by providing farmers with supplemental irrigation.

Figure 2. Suitability maps for Arabica coffee were developed jointly with DAPA team at CIAT with data from national partners across the East African region.
Figure 2. Suitability maps for Arabica coffee were developed jointly with DAPA team at CIAT with data from national partners across the East African region.

Climate Change, Agriculture, and Food Security (CCAFS)
Climate change is an immediate and unprecedented threat to the livelihoods and food security of hundreds of millions of people who depend on small-scale agriculture. To overcome these threats, the CGIAR and Earth System Science Partnership have united through CCAFS, a strategic ten-year partnership. Farmers, policymakers, donors, and other stakeholders are strongly involved to integrate end-user knowledge and needs. Synergies and tradeoffs between climate change, agriculture, and food security are studied to promote more adaptable and resilient agriculture and food systems. CCAFS is structured around four thematic research areas: (Theme 1) Adaptation to Progressive Climate Change, (Theme 2) Adaptation through Managing Climate Risk, (Theme 3) Pro-poor Climate Change Mitigation, and (Theme 4) Integration for Decision Making. Place-based research is focused on five regions: East Africa, West Africa, South Asia, Latin America, and Southeast Asia.

IITA is one of the 15 CGIAR centers involved and it particularly contributes to research on:
•    Theme 1 on crop G × E interactions. The major focus is on the IITA crops cassava, maize, soybean, yam, cowpea, and banana, but with other crops in the system being investigated as well, including horticultural crops and tree crops such as coffee and cocoa.
•    Theme 2 on plant health × climate change: IITA has a strong plant health team that is currently exploring the relationship between climate variables and major pest and disease threats, with the same crop focus as listed under G × E.
•    Theme 3 on analyzing trade-offs and synergies in climate change adaptation and mitigation in perennial-based crop systems in the humid tropics: The research focuses particularly on coffee and cocoa-based systems (see page 44 in this issue).
•    Theme 4 on communicating the results of the trade-off and carbon-footprinting analysis to the stakeholders, in particular policymakers, certification bodies, and the private sector.

Installation of an erosion control trial, Sud-Kivu, DR Congo. Photo by IITA
Installation of an erosion control trial, Sud-Kivu, DR Congo. Photo by IITA

The future of NRM
Most CRPs have moved into an implementation phase and all facilitating structures have been put in place, which is probably the most exciting change in the way of doing business within the CGIAR since its inception. From the foregoing summary, the crucial role of IITA as a whole and the NRM research areas more specifically is clear, especially for the African continent. Although IITA may have lost some of its NRM capacity over the past decade, as shown in some of the articles in this publication, much NRM innovation, strategic thinking, and practical solution development is still happening at IITA and will only be strengthened over the coming decade with the renewed investment of IITA in NRM.

Effective commercial products for farmers

Martin Jemo, m.jemo@cgiar.org, Cargele Masso, Moses Thuita, and Bernard Vanlauwe

Farmer screening soybean varieties in Kabamba, DRC. Photo by IITA
Farmer screening soybean varieties in Kabamba, DRC. Photo by IITA

Background and issues
More and more commercial products, such as biofertilizers, biopesticides, and chemical agro-inputs, are being sold to smallholder farmers in sub-Saharan Africa (SSA). However, their quality and efficacy, especially for the microbiological products, are not properly evaluated before they are commercialized, because regulations are lacking or inadequate. There is a crucial need to implement appropriate regulatory mechanisms.

When microbiological products are used as directed, they are generally more environmentally friendly than synthetic fertilizers. Also, they mainly improve soil fertility by either biological nitrogen fixation (BNF) (rhizobium inoculants) or by increasing the availability or uptake of plant nutrients already in the soil (e.g., phosphorus- solubilizing Pseudomonas putida). Unlike microbiological products, synthetic fertilizers N and P chemical fertilizers) are sometimes associated with nutrient loss to the environment causing greenhouse gas emissions or eutrophication. Hence, one of benefits of using microbiological products in integrated soil fertility management (ISFM) is to preserve the natural resource from degradation, while sustaining adequate crop production.

The goal of the Commercial Products (COMPRO-II) project is therefore to improve crop yields, improve food security, and minimize the negative impacts of bad or inadequate agricultural practices on the environment.

Figure 1. Screening framework of commercial products in Ethiopia, Kenya, and Nigeria under COMPRO-I.
Figure 1. Screening framework of commercial products in Ethiopia, Kenya, and Nigeria under COMPRO-I.

The project is built on public-private partnerships to develop effective laws and regulations for biofertilizers and other agro-inputs in SSA. It is expected that the large-scale impact of this project will be a significant reduction of inefficacious agro-inputs in the marketplace, resulting in improved crop yields.

Product screening
Products evaluated under the COMPRO project are grouped into three categories: I: rhizobium inoculants, II: other microbial inoculants, and III: non-microbiological products. However, COMPRO-II mainly focuses on categories I and II.

The product evaluation has three key steps: laboratory/greenhouse testing, field testing, and the application of appropriate ISFM (Fig. 1). An additional step consists of the scaling up of the most  promising products retained after the three key steps.

Overview of COMPRO-I results
Over 100 commercial products from the three categories were evaluated under field conditions in Kenya, Nigeria, and Ethiopia from 2009 to 2011 in the first phase of the project (COMPRO-I). A significant economic benefit to farmers was found for only a few products (Table 1). On average, the benefit–cost ratio (BCR) for rhizobium inoculants in soybean was found to be US$4.1/dollar and maize seeds coated with plant nutrients resulted in a BCR of $4.6/ dollar. A BCR of 2.5 is considered satisfactory for the adoption of the technology. The photo below also shows a significant growth improvement for faba bean following treatment with a rhizobium inoculant.

Table 1. Yield increase and benefit-cost ratio of selected products evaluated under various field conditions in Ethiopia, Kenya, and Nigeria.
Table 1. Yield increase and benefit-cost ratio of selected products evaluated under various field conditions in Ethiopia, Kenya, and Nigeria.

Analytical tools
A better understanding of the fate and dynamics of the strains in microbiological products after their application to the soil requires adequate analytical tools. In COMPRO-I molecular tools to detect the Mitochondrial Large Subunit (mtLSU) DNA of the isolate Glomus intraradices in commercial products (e.g., Rhizatech) was developed (Fig. 2). The yield increase following the application of Rhizatech was associated with faster root colonization by arbuscular mycorrhizal fungi (AMF) as determined by the mtLSU DNA tool.

COMPRO-II is further investigating the information provided by a certain region of AMF DNA (mtLSU) and the use of Real Time PCR approach to discriminate different species and isolates of AMF. For example, such tools will be used to determine factors that control BNF in cowpea, a crucial food crop, to develop appropriate inoculants for the benefit of smallholder farmers in Africa.

Figure 2. Electrophoresis gel showing fragments amplified with “INTRA” primers targeting ribosomal DNA of <em/>Glomus intraradices.
Figure 2. Electrophoresis gel showing fragments amplified with “INTRA” primers targeting ribosomal DNA of Glomus intraradices.

Future plans
Based on the economic analysis, a relatively low percentage of the commercial products evaluated under COMPRO-I showed a significant benefit to smallholder farmers. Hence, there is a need to implement adequate regulations to prevent the proliferation of inefficacious products in the marketplace and also to disseminate the most promising products by increasing farmers’ awareness about them. Such a goal can be reached only when adequate resources are available. COMPRO-II intends to address those issues based on the lessons learned from COMPRO-I. Scaling-up of efficacious microbiological products will not only contribute to improved crop yields, increased food security, and reduced rural poverty, but will also, when used in adequate ISFM, contribute to preventing agricultural land degradation caused by a lack of agricultural inputs or the heavy application of chemical fertilizers.

A farmer shows inputs used to get the healthy maize crop. Photo by FIPS
A farmer shows inputs used to get the healthy maize crop. Photo by FIPS

Inadequate crop production systems generally result in degraded agroecosystems and reduced crop yields, and therefore have negative impacts on NRM. Biofertilizers are considered environmentally friendly and, when properly used, contribute to improved soil fertility (e.g., BNF and phosphorus availability), and preserve natural resources. However, in SSA, many smallholder farmers are not familiar with those products, while regulations are virtually non-existent in many countries. The COMPRO project intends to address those gaps by: (1) screening commercial products including biofertilizers through a stringent scientific scrutiny, (2) communicating information on, and disseminating products proven best or promising, and promoting ISFM, (3) developing adequate regulations to ensure the safety, efficacy, and quality of commercial products, and (4) building the capacity of countries in SSA to implement and enforce such regulations.

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.

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.

Molecular diagnostic tools for plant health protection

Lava Kumar (L.kumar@cgiar.org)
Head of IITA’s Germplasm Health Unit and Virologist, Ibadan, Nigeria

Molecular tools in disease diagnosis
Rapid advancements in biotechnologies have led to the development of a myriad of molecular diagnostic tools in the past decade1. These tools, either based on the properties of nucleic acid (DNA or RNA) or proteins of the target agents, have improved the efficacy, accuracy, and speed of detection and identification of disease-causing agents and characterization of the diversity of pathogens and pests.

Researcher observing mouse hybridoma cell lines under microscope in the Virology and Molecular Diagnostics Unit, IITA, Ibadan, Nigeria. Photo by IITA.
Researcher observing mouse hybridoma cell lines under microscope in the Virology and Molecular Diagnostics Unit, IITA, Ibadan, Nigeria. Photo by IITA.
Most popular protein detection methods depend on antigen-antibody interactions. Polyclonal or monoclonal antibodies produced against the proteins of interest are used as probes to detect the target proteins by techniques such as enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, dot immunobinding assay, and a number of variants of these techniques, Meanwhile, nucleic acid-based diagnostic tools are based on the hybridization of homologous nucleotides, size of the DNA fragments generated by restriction enzyme treatment, order of nucleotide arrangement, or a combination of more than one of these approaches. Polymerase chain reaction (PCR), developed in the mid-1980s, has led to the development of several new and simplified techniques, fast established as a mainstay of applied molecular biology and molecular diagnostics.

Platform for development of molecular diagnostics
The objective of the molecular diagnostics research in IITA is to develop tools and technologies for better understanding, diagnosis, and monitoring of biological systems. This program emphasizes the development of simple and accurate tools and procedures for rapid identification of pathogens and pests affecting the food and horticultural crops in sub-Saharan Africa (SSA). Both protein and nucleic-acid based diagnostic tools have been developed against target agents (viruses, fungi, bacteria, phytoplasma, insect pests, and mycotoxins). These tools are critical to several programs on crop improvement and crop protection, including evaluation of germplasm for host resistance, breeding for pest and disease resistance, surveillance surveys, and monitoring programs.

ELISA-based diagnostics are preferred for the identification of plant viruses. It is simple, reliable, cost-effective, and easy to adopt in minimally-equipped labs. Backed with facilities for purifying proteins, and production of polyclonal and monoclonal antibodies, ELISA-based diagnostics were established for about 20 economically important viruses affecting IITA’s mandate crops in SSA (e.g., Maize streak virus, cassava mosaic begomoviruses, Cowpea mottle virus, Southern bean mosaic virus, and more). Antibodies were also produced against nonviral targets such as mycotoxins. Polyclonal antibodies produced against aflatoxin B1 were used to develop the ‘Afla-ELISA’ test for quantitative estimation of aflatoxins in maize and other commodities (see companion article on Afla-ELISA). Monoclonal antibodies are usually produced for discriminating closely related virus species or strains (e.g., African cassava mosaic virus and East African cassava mosaic virus). The production of monoclonal antibodies is expensive and tedious, but it offers the advantage of perpetual production of antibodies from mouse hybridoma cell lines. Because of this, IITA has placed increasing emphasis on producing monoclonals for all important pathogens.

PCR-based diagnostics are developed as an alternative tool or to overcome the limitations of ELISA in detecting viroids, viral satellites, and to discriminate strains and closely related species. Oligonucleotide primers have been developed based on the genomic data generated from our research programs and those available in the public database for the specific detection of targets in PCR assays. Procedures were also established to simplify PCR application. For instance, a procedure established for direct detection of viruses in leaf sap bypasses the need for nucleic extraction2. Emphasis is placed on the development of multiplex PCR assays for the simultaneous detection of more than one virus in a single reaction. A multiplex PCR method has been developed for the simultaneous detection of African cassava mosaic virus and East African cassava mosaic like-viruses responsible for cassava mosaic disease in SSA2. This test was further improved to detect cassava brown streak viruses that have emerged as a major threat to cassava in East Africa, thereby making it a one-stop test for detecting all the major viruses infecting cassava in SSA.

Similar efforts are being devised to detect all viruses infecting yam. Real-time PCR using TaqmanTM probes are being developed to quantify virus concentrations within the plants to characterize host response to virus inoculation. Presently, specific and generic diagnostic tools for the detection of almost all the pathogens that affect major food staples in SSA have been established at IITA.

Pathogen diversity and DNA barcodes
Detailed knowledge of pathogen diversity is a prerequisite to developing unambiguous diagnostic tools. Pathogen populations are characterized by sequencing the specific genes and the data generated is used to interpret origin and spread of the pathogen, taxonomy, and phylogeny. For diversity assessment, gene targets are selected based on the pathogen that comprise, ribosomal Internal Transcribed Sequence (ITS), mitochondrial cytochrome oxidase-I (COI), histone, virus coat protein, etc. This approach has been used for assessing the diversity of Colletotrichum gloeosporioides responsible for anthracnose of yam, Cercospora spp. causing gray leaf spot of maize, cassava brown streak virus, banana bunchy top virus, and several others agents. Information generated from these studies have provided valuable clues to understand the origin and drivers of spread, identification of previously uncharacterized pathogens3,4 and identification of unique markers known as “DNA barcodes” for use as genetic markers for identifying pathogens and pests5.

Workflow in development of protein biomarkers. Source M. Cilia, Cornell University.
Workflow in development of protein biomarkers. Source M. Cilia, Cornell University.
Biomarkers for insect vectors
Recently a new initiative was started in collaboration with Cornell University to identify protein biomarkers to rapidly identify variation in vectoring potential of aphid and whitefly vector populations. Diagnostic tools developed in this program will aid in better understanding the virus-vector interactions, disease epidemiology, and improved management of insect vector-borne virus diseases.

Training in application of molecular diagnostics
In addition to technology development, efforts are made to transfer technology, products, and skills to stakeholders in national research and extension services. This is done through collaborative activities and organization of training courses at regular intervals in collaboration with national organizations such as the Nigerian Institute of Science Laboratory Technology (NISLT). During the training courses, specific emphasis is placed on the application of diagnostics in monitoring and surveillance programs. Standard diagnostic protocols are compiled into a cook-book style laboratory manual6 and distributed during the training courses.

End note
Molecular diagnostics development programs in IITA consider the latest knowledge and state-of-the-art technologies in establishing simple and robust tools that are relevant to end-users, are low-cost, and conducive for adoption in minimally equipped labs. We are adding new tools, such as, loop-mediated isothermal amplification reaction (LAMP) assay and deep sequencing approaches to broaden the knowledge on pathogens occurring in our mandate crops to increase the repertoire of available tools.

Molecular diagnostic tools are routinely used in germplasm indexing, phenotypic evaluation of germplasm, disease surveillance, and monitoring programs in SSA. They are also used in collecting baseline information and monitoring shifts in pathogen and pest dynamics due to changes in agriculture systems and climate change effect. These tools are already proving useful in rapid detection and identification of new and emerging pathogens and pests [e.g., Paracoccus marginatus (papaya mealybug) in Nigeria; Phytophthora colocasiae causing taro leaf blight in Nigeria and Ghana; 16srII group phytoplasma responsible for witches’ broom disease of soybean in Southern Africa; and Banana bunchy top virus in Benin].

References
1 Benali, S., et al. 2011. Eur. J. Sci. Res. 50:110–123.
2 Alabi, O.J., et al. 2008. J. Virol. Methods 154:111–120.
3 Alabi, O.J., et al. 2010. Arch. Virol. 155:643–656.
4 Sharma, K., et al., 2010. Phytopathology 100 (6): S117.
5 Kumar, P.L. and K. Sharma. 2010. DNA barcodes for pathogens of African food crops. R4D Review 4: 51–53. www.R4DReview.org.
6 Kumar, P.L. (ed.). 2009. Methods for diagnosis of plant virus diseases: a laboratory manual. IITA, Ibadan, Nigeria. 90 pp.

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.