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Emerging Technologies to Benefit Farmers in Sub-Saharan Africa and South Asia (2009)

Chapter: 3 Plant Improvement and Protection

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Suggested Citation:"3 Plant Improvement and Protection." National Research Council. 2009. Emerging Technologies to Benefit Farmers in Sub-Saharan Africa and South Asia. Washington, DC: The National Academies Press. doi: 10.17226/12455.
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3 Plant Improvement and Protection Enhancing Crop Performance Crop performance is affected by a combination of many factors, not the least of which is the collection of genes (or variants of individual genes, referred to as alleles) that provide the plant with the potential for high yield in a given farming environment. The right genes or alleles to overcome the constraints identified in Chapter 2 and enhance desirable traits in a crop are brought together by plant breeding, a complex process that, broadly defined, uses all the tools of modern plant science, including agronomy, field trials, propagation, tissue culture, genomics, molecular biology, biochem- istry, and plant physiology. This chapter describes existing and evolving tools for improving and protecting crops. The first and largest part of the chapter focuses on plant-based applications; the second part addresses plant protection using biological control methods. For many crop species, sources of germplasm that are able to overcome particular constraints have been identified, and conventional breeding tech- niques can be used to bring together the desired genes or alleles (Pingali, 2001). Within the range of germplasm available to breeders, crops contain alleles that can improve performance with respect to a variety of traits, but in many instances they do not. This is where the potential for genetic engineering of crop plants can make a tremendous contribution. Novel genes for improving a crop can come from plant, animal, or bacterial spe- cies, and molecular techniques are used to introduce them into a candidate crop. Once they are introduced into a plant, conventional plant breeding approaches are used to incorporate them into the local, elite germplasm. 71

72 Emerging Technologies to Benefit Farmers Conventional and transgenic approaches to enhance crop performance are complementary and rely on many of the same molecular and informational tools. Box 3-1 contains a list of plant traits of which variants are selected by breeders (sometimes inadvertently) as targets in the process of creating improved plants for various environments. Many of the traits are linked by biochemical interactions that influence the expression and control of gene products. Sometimes the genes controlling the different traits are ge- BOX 3-1 Examples of Traits Targeted for Improvement •  rchitecture—height, number of leaves, tillers, branches, leaf angle, number A of flowers and seeds, seed size, root structure, surface area. • Optimal planting density. • Flowering time and photoperiod responses. •  rowth rates and regulation of hormones: brassinolide, auxins, gibberellins, G cytokinins, ethylene. • Growth responses to light quality and quantity. •  hotosynthesis rates and overall carbon fixing during growing season, chlo- P roplast number and positioning, C3 vs. C4 metabolism, pathway regulation. • Heterosis (hybrid vigor) and male sterility for hybrid production. • Fertility, inbreeding and outbreeding. •  itrogen and phosphorus uptake, use efficiency, translocation, storage, reduc- N tion, portioning between plant parts. •  ater use efficiency: uptake, storage, transpiration rates, loss, tolerance of W chronic drought and transient drought. • Heat or cold shock and sustained tolerance to heat, cold, freezing. • Seed germination in cold. • Flooding tolerance. • Oxidative stress tolerance. • Heavy-metal and salt tolerance. • Biosynthesis of key metabolites. • Nutritional composition—seeds, roots, leaves, fruits, stems. • Digestibility (by humans and animals). • Root endosymbionts. • Resistance to viral, fungal, and bacterial pathogens. • Resistance to weeds and to herbicides that control them. • Resistance to insects and other pests and predators.

Plant Improvement and Protection 73 netically linked and do not undergo recombination in meiosis. Failure to understand these associations leads to inefficiency in the breeding process and to poor crop performance. These relationships exist even when a new, transgenic trait is introduced into a plant genome, which emphasizes the need to continue improving germplasm even when transgenic solutions are available for some traits. It is beyond the scope of this report to describe a strategy for every trait that would be worth targeting. Examples of opportunities with potentially high returns are discussed throughout the chapter, which focuses on oppor- tunities to overcome some of the major problems that constrain agricultural productivity in sub-Saharan Africa (SSA) and South Asia (SA). Essential Features of Breeding Programs Because the success of every trait modification project depends on the competence of breeding programs, it is worth drawing attention to the quality of the breeding process itself. In particular, two essential features of modern breeding programs should be emphasized. First, successful crop improvement is based on a foundation of knowledge that informs all the intellectual and physical efforts of plant breeders in the laboratory and in the field. The different types of information needed are likely to be gener- ated by a variety of sources, so the task of plant breeders is to draw the information together as the basis of a breeding strategy. With relevant information, breeders can consider options for genetic and operational tactics to resist disease, weed, and pest damage; to enhance yield traits; and to begin a science-based, comprehensive breeding program to generate candidate germplasm. Among the types of knowledge important to plant breeders are the following: • An understanding of the appropriate crop for breeding and its fundamental genetics. This knowledge comes from extensive analysis of the needs of specific human societies (including farmers and consumers) and of the relevant production constraints on a crop that have the potential to be genetically manipulated. • An understanding of the markets for and uses of crops and the types of improvements that can add value to the crops. • Knowledge of the genetic and phenotypic diversity in the available crop germplasm (enhanced by similar information on other crops). • Knowledge of the biology of relevant pests, weeds, diseases, and stresses that routinely limit yield in the crop. • Knowledge about the specific processes that result in yield loss in farmers’ fields.

74 Emerging Technologies to Benefit Farmers The second feature of a successful breeding program involves under- standing that in evaluating germplasm, trials and selections take place in environments similar to those in which the crop will be grown. Therefore, crop yield trials in these regions are essential, requiring trained local man- power. The international agricultural research centers of the Consultative Group on International Agricultural Research have endeavored to establish consortia with national programs to achieve this testing, but an expansion of the effort is needed. Trials can be carried out under favorable conditions to estimate yield potential, but such conditions are not usually encountered area-wide. Additional realistic trials need to be conducted to select toler- ance traits relevant to the major constraints under conditions encountered by small-scale farmers. Similarly, a fundamental tenet of plant breeding is the ability to assay the trait in question. For example, during breeding and selection, plants have to be subjected to the most common strains of pests, weeds, and diseases that will challenge them in the farmer’s field. That is not a simple task, because given that pests, weeds, and diseases are not prevalent every year, breeding and selection processes need to be coupled to tests for tol- erance and sensitivity to the specific strains of indigenous or potentially indigenous pests, weeds, and diseases. Local and emerging diseases need to be precisely identified for each crop; the involvement of local farmers in the selections and tests could increase the likelihood that the final products will be adopted, and additional local knowledge can be incorporated into the selection processes. The establishment and scale-up of modern plant breeding programs in SSA and SA should have high priority in any organization looking to improve agricultural productivity. Plant breeding is rapidly evolving as an integrative technology supported by increasingly powerful molecular tools. Most of the expertise and knowledge needed to carry out this work is already available somewhere in the world. The message to the interna- tional development community is that crop improvement ultimately needs to be understood as a local and regional effort that is assisted by tools and knowledge developed by a broader community. Existing Tools for Conventional Plant Improvement Annotated Sequences of Crop and Model Species for Comparative Genomics Some of the knowledge that plant breeders need to improve crops in SSA and SA already exists or is rapidly being generated. The sequences of the genomes of corn, sorghum, rice, and poplar have been or soon will be published. The cassava genome is currently being sequenced by the U.S. Department of Energy Joint Genome Institute. The genomes of tomato,

Plant Improvement and Protection 75 the legume Medicago truncatula (barrel clover), and many other species are in line to be started or completed, although more attention to impor- tant legumes such as the common bean, cowpea, and pigeon pea, as well as other crops important to the poor such as species of Musa (e.g., banana and plantain) would certainly be welcomed. Nevertheless, the complete sequences of several genotypes of many additional crops will be known in 5 years; nearly every gene will be identified, variants for many important genes found, and the most relevant genes’ associations with traits estab- lished. The patterns of expression of co-regulated genes will become linked to phenotypes (the expression of the collection of genes that make up a trait in a given environment). Easily scorable genetic markers for every small chromosomal segment of much of the relevant germplasm will be known, and determinations of which chromosomal variants to select for in different environments will begin to emerge. All that will usher in a new platform for genomics-based breeding in which the needed genomes exist in the crop or in interbreeding relatives. Transgenic technologies will be needed to transfer the genes from other species where that is not the case. Two plants in particular are important models of the major species grown in SSA and SA. One is Arabidopsis, the most-studied reference plant; over the last 20 years, a detailed understanding of the molecular, biochemical, and cellular basis of pathways and circuits in Arabidopsis has been developed, and it will remain the leading source of knowledge on the biological systems of plant traits in the coming decade. The other is rice, the model plant of the grass species, including maize, wheat, pearl millet, sorghum, and others. Progress in understanding the biology of rice traits will be slower than in that of Arabidopsis, but it will be more directly rel- evant to cereal crops in the developing world. Research tools and genetic stocks available to address the fundamental questions concerning potential constraints on rice yield are rapidly increasing and are being used by a growing consortium of scientists around the world. Moreover, because of their common ancestry, the grasses have retained the same general order of genes along chromosomal segments (synteny), so mapping of genes on chromosomes of one species can be helped enormously by knowledge of a reference genome, such as that of rice or maize (Bennetzen and Ma, 2003). Diversity in gene order sometimes exists between these genomes over short distances, but this does not eliminate the value of synteny in aiding com- parisons between crop genomes. It also helps in finding truly orthologous (similar) genes between species. DNA Markers DNA markers—sequences of DNA shown to be associated with par- ticular genes or traits—have great potential to assist plant breeders (Jena et al., 2006; Steele et al., 2006), but for various reasons they have been

76 Emerging Technologies to Benefit Farmers underused in breeding programs. The use of qualitative phenotypic infor- mation with markers to determine the optimal crosses and offspring can shorten the cycle of crop improvement from 5 to 2 years (Jannink et al., 2001; Arbelbide and Bernardo, 2006). Methods for assaying genetic vari- ants are readily available and improving each year. When the contribution of a variant gene to a trait is known, the variant can be used as a marker of the trait. A candidate plant for breeding can be identified by the pres- ence of a marker in its genome without the need to test it for its phenotypic expression. That can help in choosing diverse parents for specific traits; in reducing the number of breeding generations by making it possible to select homozygotes more efficiently; in accelerating backcrossing of a trait to an elite parent, especially when the desired trait is recessive; and in selecting desirable progeny while rejecting poorer genotypes without the need for complex assays, such as assays of tolerance to diseases. The use of molecular markers has highlighted the importance of genes from wild relatives for crop improvement (Tanksley and McCouch, 1997; Koornneef et al., 2004). As evidenced by recent work on tomato, the results of introgressing ancestral genes can sometimes be spectacular (Frydman et al., 2004). Another example of the richness that diversity can provide is New Rice for Africa, NERICA. African farmers are showing enthusiasm for these new inter-specific hybrids that combine the best of Asian and African rices (Jones et al., 1997). Better knowledge of the genetic diversity of indig- enous tree species, particularly those of central Africa, could be applied to forest improvement (Juma and Serageldin, 2007). Genetic markers that link DNA sequences to traits are increasingly available in rice, maize, cassava, cowpea, wheat, and sorghum, and these crops are becoming better understood genetically (Buckler and Thornberry, 2002; McCouch et al., 2002; Somers et al., 2004; Duputie et al., 2007; Huang and Wu, 2007; Timko, 2007). Box 3-2 provides one example of the value of such markers. In general, however, tropical forage plants have received little attention from molecular and conventional plant breeders with a few notable excep- tions: alfalfa (which is grown in some tropical highlands), Brachiaria spp., Pennisetum purpureum (elephant or napier grass), and Panicum maximum (Guinea, colonial, or Tanganyika grass) have been studied (Jank et al., 2005). Characterization of forage traits that need improvement and studies of genetic markers for those traits have just begun, and temperate forage still receives far more attention than that grown in the tropics (Spangenberg et al., 2005; Smith et al., 2007). The burgeoning interest in biofuels, includ- ing use of switchgrass, should complement and accelerate our understand- ing of processes related to those occurring in forage digestion by ruminants. However, the collections of germplasm of tropical forage are poorly funded, and loss of current accessions (separate populations) is a distinct threat.

Plant Improvement and Protection 77 BOX 3-2 Molecular Breeding and Transgenic Approaches Can Be Combined to Offer New Approaches to Crop Improvement A recent publication offers a striking example of how use of the tools of molec- ular breeding can be coupled with transgenic technologies for crop improvement. Using knowledge gained from extensive breeding efforts that had identified a number of quantitative trait loci (QTLs) in rice that related to number of grains per panicle, plant height, and heading date, Xue et al. (2008) used map-based cloning to identify the gene underlying one such major QTL. The gene was identified as a CCT domain protein that plays a key role in regulating photoperiod-controlled flowering and may also control a number of other functions in growth and dif- ferentiation. The superior allele for the gene identified by this technique was then inserted and overexpressed in a recipient rice cultivar, leading to dramatic altera- tion in yield potential, plant height, and heading date. This information can now allow breeders to introgress this trait into other locally adapted rice cultivars. Mutation Breeding and Mutant Analysis TILLING (targeting induced local lesions in genomes) is a method whereby natural or induced mutations in known genes are created in large populations of plants and the populations are screened for the mutation with sensitive molecular biology methods (Henikoff et al., 2004). When plants with a mutation in a selected gene are found, their phenotype can be studied in detail, and relationships between the gene and a trait can be assessed. Most mutations are not beneficial, but if mutating a gene leads to a specific phenotype, its relationship to a trait can be inferred. In the rare cases in which a mutation is beneficial, the approach can be used to identify useful mutant alleles that can be introduced into a crop plant by conventional breeding (see Box 3-3); this constitutes a nontransgenic method for altering deleterious traits or modifying biochemical pathways. Many approaches to mutant analysis and control of gene expression have been used in Arabidopsis and rice. Making mutant analysis relevant to crops in SSA and SA will require high-throughput assessment of the phe- notypic consequences of overexpression, underexpression, or mutation of candidate genes in the crops as identified in functional genomics studies of Arabidopsis and rice.

78 Emerging Technologies to Benefit Farmers BOX 3-3 Nontransgenic Herbicide Resistance in Maize for Striga Control Breeding for Striga resistance has been somewhat successful in sorghum, a plant that is native to Africa with different strains and wild relatives whose germ- plasm carries modicums of resistance to Striga (Ejeta, 2005; Ejeta and Gressel, 2007). If and when such genes are isolated, they might be transgenically trans- ferred to other crops such as maize. Recently, a non-transgenic approach has been developed for maize that is showing good promise in the fields of Western Kenya. Unlike sorghum, maize did not co-evolve with Striga and is expected to have fewer genes for resistance, and all breeding efforts (of which there have been many over 3 decades) have given rise to lines that at best work in certain locales but not others. At present, the only technology that seems to work over large areas is mutant-based resistance to systemic herbicides, which has been back-crossed into local elite germplasm (Kanampiu et al., 2003). The herbicide is applied to the seed and requires far less chemical (one-tenth) than is typi- cally sprayed, and this does not require spray equipment. Because the herbicide remains in the maize root zone, legumes can be interplanted and not affected (Kanampiu et al., 2003). This technology is also appropriate for other crops af- fected by Striga. Three groups are generating mutant sorghums resistant to the same groups of herbicides. With the mutant sorghums there is the inevitability that the resistance gene will flow to major sorghum weeds, namely shattercane and Sorghum halepense (Ejeta, 2005). This will not give an advantage to those weeds as long as only seed treatments of herbicide are used and the seed is certified to be weed-free. exisTing and evolving Tools for Conventional and Transgenic Approaches to Plant Improvement Studies of the commercialization of discoveries in many disciplines often reveal that 2 decades pass before consumers see the results from discoveries translated into products. In plant innovation, the timescales are often especially long because the long generation times of plants and the requirement to test an innovation in multiple versions of a plant in multiple environments add years to the process. In addition, public-sector labora- tories responsible for plant breeding in SSA and SA rarely have the means to adopt new technologies rapidly and on a sufficient scale to achieve the high impact that is possible. Using the pace of a multinational plant breeding company as a bench- mark, obtaining a research finding and testing it in a model plant might

Plant Improvement and Protection 79 require 5 years, and deploying and optimizing a trait in elite germplasm for SSA and SA another 5 to 10 years. Production, testing, and distribution of a crop for consumers could then take another 5 to 10 years, especially if the crop is transgenic and therefore has a longer regulatory testing phase under current regulatory regimes. The development of such transgenic technolo- gies would need to be done in conjunction with the development of crops that are appropriate for SSA and SA. This section highlights frontier, paradigm-changing technologies that show promise for agriculture but need further development. Creative, world-class research will be needed to move them into practice if they are to be incorporated into plant breeding programs and production agriculture. And the technologies will require education and training for users, from the breeder to the farmer and consumer. Technologies for Rapid Sequencing and Annotation of Crops of Sub-Saharan Africa and South Asia The most fundamental tool in modern plant breeding is a complete and annotated genome sequence of a crop of interest coupled with the ability to probe the DNA of selected germplasm to look for favorable gene combina- tions. Because that tool does not exist for many of the crops of importance to farmers in SSA and SA, the sequencing of the genomes of these crops is identified as an emerging and essential tool for plant improvement. For example, temperate maize (or corn), the focus of U.S. studies, is different from tropical maize. How? And what genes are involved in the dif- ferences? U.S. and European crop improvement programs in both the public and the private sectors tend to target temperate crops, so understanding how many genes control the temperate vs. tropical phenotype would seem to have value in connecting the “northern” efforts with the “southern” and in looking at the effects of global warming on agriculture (for example, for maize, see CIMMYT, 2007). High-quality reference sequences and genome annotations of all the relevant major crops in SSA and SA can be built on the sequences and annotations of rice and sorghum already available, the emerging sequence of maize, and the reference sequence of Arabidopsis. Breeders in SSA and SA also need resequencing capacity to complement their efforts in assess- ing and understanding the genetic diversity in the available germplasm of these major crops. Much of the sequencing work could be accomplished wherever there are adequate facilities and staff. A number of radically new sequencing technologies have become com- mercially available within the last few years and have resulted in a dramatic increase in the speed of DNA sequencing and a decrease in the cost. State- of-the-art machines now generate up to 500 million bases per day. At least

80 Emerging Technologies to Benefit Farmers six companies are in a race to deliver “a complete human genome sequence for $1,000.” The agriculture sector has an opportunity to capitalize on this race. Gaining help from world-class sequencing centers that are likely to test and purchase machines is essential for securing early opportunities for application to the germplasm of SSA and SA. Large efforts are also being made in developed countries to obtain the sequences of major insect pests, pathogens, and weeds. These are neces- sary for determining weak points that might be targeted and determining whether to try to find new insecticides, fungicides, and herbicides or to try to use RNA interference (RNAi) or other technologies for control. Such in- formation is not being garnered for SSA-specific and SA-specific constraints, so these areas are at a disadvantage. Information Technology and Computational Biology One of the single most important activities for improving breeding ef- forts across SSA and SA will be in unifying available information, especially from national programs. This will involve data curation, germplasm geno- typing, and breeding value estimates based on markers. Crop varieties will need to be evaluated in tens to hundreds of locations to make rapid progress so that a wide range of environmental fluctuations are experienced in a single year. When DNA is sequenced quickly and at low cost, it probably will no longer be a bottleneck in plant science and breeding. However, man- aging all the data generated on each crop to create the reference genome sequences and to define genetic diversity with a high degree of accuracy will require substantial attention from researchers in the biological sciences and information technology (IT). There is tremendous opportunity to apply 21st century bioinformatics —which merges techniques from applied mathematics, informatics, sta- tistics, computer science, artificial intelligence, chemistry, and biochem- istry—for effective plant breeding. In many regards, breeding efforts in developing countries are not unified and trials are not well replicated, and much of the agricultural efforts are similar to in situ breeding efforts of the late-19th and early-20th centuries in the United States that provided almost no yield increase for maize. Therefore the potential to leapfrog and breed more effectively is great if researchers in SSA and SA are able to implement some of these existing techniques for plants with user-friendly computer programs to access and use genomic information. IT and software innova- tions will be needed to enable the agriculturally oriented programs that can help breeders to profit from knowing the sequence and position of all the genes in the chromosomes of SSA and SA species. Accurate and easy an- notation of all the genes in a genome sequence is still beyond the ability of the scientific community. As soil, climate, weather, and remote sensing data

Plant Improvement and Protection 81 and models continue to advance, these parameters combined with genomics and informatics could be especially helpful for adapting crops for specific environments. Computational biology and IT should be a special focus of an effort to bring the power of data acquisition to the practice of plant breeding and crop production. Technologies for Determining Genetic Variation in Key Crops Plant improvement is based on and necessarily exploits genetic varia- tion. Thus, being able to characterize the variation in every gene in the plants of a breeding nursery can bring the most powerful knowledge to the breeder. The breeder’s dream would be to know and understand the genetic diversity of equivalent chromosomal segments in the crop germplasm. Such knowledge would revolutionize the ability to pick parents successfully and select progeny more successfully. However, few breeders recognize the po- tential importance of that information and are content to focus on “good x good” crosses, ignoring the major benefits that might be hidden in other, less adapted germplasm. Equivalent chromosomal segments evolve independently in different populations but can be brought together in new combinations in breeding programs. It is desirable to know how many substantially different versions (haplotypes) of each chromosomal segment are present in the germplasm of a species and what the differences are. Answers to such questions are provided initially by the use of markers that measure sequence differences in chromosomal DNA (McCouch et al., 1997; Mohan et al., 1997; Bernado and Yu, 2007). The commercial technologies for using markers are advancing rapidly, to the point where tens of thousands of data points can be gathered in a day. The technologies for measuring polymorphisms are many and are evolving rapidly in synchrony with the DNA technologies described above because high-throughput sequencing technologies are excellent for revealing sequence differences. With efficient sequencing technologies, it is possible to reveal variants of the same gene or allele between hybrids or different acces- sions. It can be accomplished for thousands of genes at a time in genomic DNA, in libraries of complementary DNA (cDNA), or in selected regions of the genome. With high-throughput sequencing technologies, fragments of copies of messenger RNAs can be sequenced to reveal rarely expressed genes. When the fragments of genes are assembled, they can be aligned with genomic DNA sequences to define the correct gene structure. By sequencing copies of mRNAs from different accessions, one can identify single nucleo- tide polymorphisms (SNPs) between allelic genes and use them as markers. That is now the fastest way to obtain polymorphic markers (Barbazuk et al., 2007; Emrich et al., 2007), and crops in SSA and SA could be brought

82 Emerging Technologies to Benefit Farmers to that state of knowledge rapidly. Other technologies rely on hybridization of DNA or RNA to reference DNA sequences that have been attached to solid materials and then analysis of the differences between test and refer- ence sequences (Kirst et al., 2006). Separation of polymerase-chain-reaction (PCR)-amplified DNA sequences from different genomes and fractionation to reveal size differences, which are often due to variation in the number of short repeats in or around a gene, is another commonly used technique (McCouch et al., 2002). The technologies discussed above, such as DNA sequencing, are being driven by the need to describe associations between genetic characteristics of humans and their healthcare needs. They can also be used by the plant science community, but they will need to be applied on a much greater scale for the relevant germplasm and breeding programs of SSA and SA. Plant breeding depends on assessing large numbers of progeny, and large-scale applications are essential. A major goal should be to have haplotype maps (see “Analysis of Gene-Trait Associations” below) globally available for all the germplasm of a crop, easily accessible in databases, and with full phe- notypic descriptions and details of the phenotyping protocols and of all the plant accessions that have each of the haplotypes. That will enable many more scientists to become involved with the problems and opportunities in crop improvement. Just as with the DNA sequencing described above, the generation of large datasets needs to go hand in hand with IT and software innovations and with the development of user-friendly databases to enable breeders to obtain the benefits of all new information. Achieving that goal will require the help of world IT experts, most of whom work outside the agricultural community. Proteomics DNA sequences alone do not provide sufficient information on how genetic information is transcribed or translated into functional proteins. Proteomics is emerging as a powerful method for annotating structural and functional aspects of the genome, and it complements DNA-based technolo- gies. Direct measurement of protein identity and quantity was not possible on a genome-wide scale until recently, but advances in chromatography, electrospray ionization of peptides, tandem mass spectrometry, bioinfor- matics, and computer architecture have made it possible. A method called multidimensional protein identification technology (MudPIT), supplemented with a conventional two-dimensional gel ap- proach for identifying proteins, was used to create the most complete de- scription of a plant proteome; it identified more than 2,400 proteins in rice (Washburn et al., 2001; Koller et al., 2002). The method was later used to

Plant Improvement and Protection 83 demonstrate that the genomic complement of proteins of a plant species on which little or no DNA sequence information is available can be identified by relying on the expanding sequence database from other plant genomes. Nearly 200 proteins of wheat amyloplasts were directly identified with that method (Andon et al., 2002). It is now possible to observe 8,000 or more proteins of whole plants with highly reproducible quantitative comparisons between samples. Peptide mass spectrometry can reveal the exon-intron composition of genes, including which splice isoforms are present, amino acid sequence polymorphisms, and post-translational modifications. Proteins and their subcellular compartments can be identified by using mass spectrometry in conjunction with cell fractionation. These kinds of information cannot be obtained reliably from genomic DNA sequences alone. Peptide mass spectrometry analysis of a single human cell line showed that hundreds of human gene models were wrong. The data allowed the previous models to be corrected and validated gene predictions regarding hundreds more hypothetical proteins. In all, peptides from 39,000 exons and 11,000 exon- spanning junctions were observed (Tanner et al., 2007a,b); this made clear how much value genome-wide proteomics can bring to genome annotation, including cases in which little or no supplementary information, such as cDNA sequences, is available. Peptide mass spectrometry also permits the identification of single amino acid polymorphisms arising from allelic DNA sequence differences. About 20,000 peptides can be routinely observed in a plant sample analy- sis, and this facilitates the detection of thousands of genetic markers. In the data on the human cell line described above, more than 300 known SNPs were confirmed as single amino acid polymorphisms. The level of polymorphism in plant breeding populations would most likely be greater. It is possible that antibodies that discriminate between single amino acid polymorphisms could be used to enable high-throughput, low-cost enzyme- linked immunosorbent assay (ELISA) for genotyping breeding populations. By combining discovery of polymorphic peptides with mass spectrometry and low-cost, conventional ELISA detection, it should be possible to deploy robust assay systems that accelerate breeding. Peptide mass spectrometry also has recently enabled the genome-wide discovery of all post-translational modifications (Tanner et al., 2007b). Fractionation methods have recently been combined with fluorescence- tagged proteins to assign selected proteins to subcellular locations. That approach is low-throughput, therefore the locations of very few proteins are known. Combining fractionation methods with peptide mass spectrometry has the potential to establish the subcellular locations of all proteins. Many proteins change locations during their function cycle. For example, protein products of plant disease-resistance genes migrate from the cytoplasm,

84 Emerging Technologies to Benefit Farmers where they are activated by pathogen attack, to the nucleus, where they induce a defense response (Wirthmueller et al., 2007). Every case of reloca- tion has been discovered after years of painstaking research. Peptide mass spectrometry has the potential to reveal all the relocation dynamics of the proteome and to associate changes with performance traits. Despite the exceptional opportunities for discovery in and practical benefits of peptide mass spectrometry research, there is little grant funding to support it and few people have the training required to do it. Systems Biology for Analysis of Complex Traits Breeders are well aware that key traits—such as drought tolerance and durable resistance to diseases and pests—are complex and involve many genetic loci, and it is a major goal in biology to have an integrated understanding of these dynamic, complex traits, their regulation, and how they create form and function. Gaining that understanding requires many observations and the development of computer-based simulations of the processes. These tools provide new ways of designing experiments to test hypotheses. Besides static models of how plants function, dynamic models of the developmental programs of plants can build in the plant’s responses to environments. Such progress will probably first come from Arabidopsis and other species such as yeast, Caenorhabditis elegans, and bacteria. For example, scientists studying the yeast Saccharomyces cerevisiae have con- structed a predictive mathematical model for specific signaling pathways and use oscillatory stimuli as a surrogate for environmental conditions to demonstrate how networks of proteins and genes are engaged by a living system to control physiological behavior (Mettatal et al., 2008). That physiological behavior will be expressed not only in the form of gene products (proteins) but also in the dynamic levels of small molecules (metabolites) produced as biochemical pathways ramp up and down ac- cording to different environment conditions, such as nutrient levels or temperature. Metabolomics, the high-throughput, comprehensive analysis of metabolites in the tissues of an entire sample (or even an entire plant), will provide important data for the models of biochemical pathways used in systems biology (Allwood et al., 2008). A systems approach can also facilitate better strategies to manipulate multiple transcription factors that regulate the biochemical pathways that control complex traits. Recent very promising results demonstrate that al- tering the expression of even one such master regulator of a single pathway can alter the responses of plants to drought in maize (Nelson et al., 2007), resistance to disease in rice (Zhang et al., 2008), or control of developmen- tal programs that control architecture, plant growth rates, and yield poten- tial in rice (Xue et al., 2008). One can only anticipate that understanding

Plant Improvement and Protection 85 the interactions among various transcription factors will lead to even better approaches for the control of complex traits (Century et al., 2008). Work on Arabidopsis in the next 5 years will encompass the sequenc- ing of its many variants, mapping of quantitative trait loci (QTL), and the discovery of the genes behind the QTLs. The expression patterns of every gene, the role of microRNAs and small RNAs, and the levels of mRNAs in development and environmental responses will be understood (Lu et al., 2006; Maher et al., 2006). There will also be greater knowledge about the epigenetic control of genes and their processes during development and in different genetic backgrounds, about biochemical pathways and the relationships between metabolites and physiological states, and about the control of growth by multiple factors, especially hormones. The genetic basis of some examples of hybrid vigor (Springer and Stupar, 2007) will probably be understood. Although the information will come first from noncrop species, it will have enormous value for crops in SSA and SA (see Box 3-4). The information will reveal the genetic, biochemical, and physi- ological basis of traits at the cell, tissue, organ, and whole plant levels. It will hopefully lead to new insight into how to improve the traits for specific crops and purposes. Analysis of Gene-Trait Associations Geneticists have made great progress in locating, on genetic maps, the loci that are linked to particular traits (Mohan et al., 1997; Steele et al., 2006). The use of polymorphic genetic markers covering all the chromo- somal sets allows the linkage of a marker in a chromosomal segment to a trait in populations in which the trait is segregating. The existence of huge datasets of mapped sequence polymorphisms means that finding genetic markers of traits is not rate-limiting. What is rate-limiting is the measurement of phenotypes. In plant breed- ing, the phenotype needs to be ascertained for hundreds or thousands of progeny from a large number of crosses for each species to reveal which loci move together in heritable associations. It is also desirable for the strength of the phenotypes to be measured in multiple environments. To measure traits that affect a disease, the plants being tested must be exposed to the disease—an enormous task. An alternative to that laborious process is to compare markers and traits in as large a number of unrelated accessions of a crop as possible (Yu and Buckler, 2006; Ersoz et al., 2007). It might be possible to infer a close and possibly causal relationship between a marker (or gene) and a trait by seeing whether they are inherited together at a higher frequency than would occur randomly. The process of “association mapping” and the development of haplotype maps are being studied in maize in detail (Yu and

86 Emerging Technologies to Benefit Farmers BOX 3-4 Understanding Lignin Synthesis for Improving Tropical Forage In many cases in SSA and SA, forage-fed animals lack nutrients to sustain growth or lactation and are struggling to meet their basal requirements The C4 grasses (so named for the metabolic pathway used to fix carbon dioxide) that typi- cally predominate in the tropics are about 15 percentage units less digestible than are temperate C3 grasses (Van Soest, 1994) and have low forage energy. In C3 grasses, highly digestible mesophyll cells accumulate during development; in C4 grasses, parenchymatous bundle-sheath cells form between the vascular bundles and reduce digestibility (Wilson and Hatfield, 1997). Differences in the hemicel- lulose and lignin fractions of C4 grasses and C3 legumes also affect digestibility. The lignin-hemicellulose cross-linking (ester linkages in grasses vs. ether bonds in legumes), lignin monomer composition, and functional groups render grass lignin more soluble in alkali than lignin in legumes (Van Soest, 1994). In addition to better understanding of cell-wall chemistry in model plants, including maize (a C4 grass), progress has been made in understanding the genes that control lignin biosynthesis and their regulation (Ralph et al., 2004), and lignin content has been modified and digestion improved, not only in the model plant tobacco (Spangen- berg, 2005) but also in alfalfa (Chen and Dixon, 2007) through engineering of the expression of key enzymes. The next steps involve application of those results to the understudied tropical forage and genomic research on pasture species that are well-adapted to stressful environments (drought, low soil fertility, and salinity) to identify novel genes. Using a systems biology approach to understand plant chemistry and lignin synthesis could help plant breeding programs to improve the nutritional value of forage and would also complement ongoing efforts to enhance the accessibility of cellulosic residues in crops targeted for biofuels. Buckler, 2006) but could be promoted for many more crops important to the developing world. QTL analysis uses statistical frequencies of alleles to suggest the strength of relationships between loci and quantitative or continuous traits. QTLs in rice, maize, and other crops have been studied extensively (Young, 1996; Steele et al., 2006; Szalma et al., 2007). Many QTLs have been mapped, and candidate genes responsible for them are being nominated. All this informa- tion on multiple species can be brought together to establish hypotheses for one crop species using results on another. In the field of functional genomics, many gene-trait associations have been established by adding new genes, or upregulating or downregulating

Plant Improvement and Protection 87 existing genes in plants and examining their effects on a trait. In the course of such studies, genetic changes that cause changes in a trait imply that a transgene controls a step that is connected with regulation of the trait. Such genes are likely to be especially interesting and of special significance in plant breeding. Because the functions of new genes are being discovered rapidly in model plants, it seems important to promote efforts to carry out surveillance of the results with an eye to identifying and testing in crop plants the genes that may have particular relevance for international agriculture. The work involved in establishing the genetic variation that is present in a crop and which genes contribute to which traits is massive. It will be a huge undertaking but will gradually transform plant breeding into a more directed, efficient process driven by technologies different from and more powerful than those of today. Hyperspectral Imaging and Digital Capture Since the mid-1980s, various groups have been developing remote sensing technologies capable of collecting hyperspectral images from high- altitude aircraft and orbiting platforms. Hyperspectral images provide high-resolution spatial and spectral data (in the visible and infrared light spectrum) that can discriminate small differences between objects, such as crops and weeds. The images are collected by charge-coupled devices (CCDs) that have been developed to permit high-resolution imaging with broad wavelength capability and very fast signal readout. Typical CCDs can record 200-plus spectral channels for each pixel over a range of 0.4 to 2.5 μm. Differences between the spectral reflectance curves of plant parts can distinguish differences in species and genotype, surface structure, and chemical characteristics (Liew et al., 2008). In addition, light penetrates leaves to a depth of some 50 to 200 μm, so spectra of internal structure and chemistry can also be gained, reflecting changes during plant development, during senescence, and in response to stress, disease, variation in nutrient- use efficiency, water-use efficiency, and photosynthetic activity. Just as it is possible to make remote hyperspectral measurements and compare them reliably with reference spectra in the field, it is also possible to develop automated spectral tools coupled with methods of multivari- ate spectral calibration to assess the physiological state and some internal chemical composition characteristics—those of seeds, leaves, fruits, and so on—by direct spectral measurements. More research is needed to learn how to understand the variation in a subject plant’s spectra during the day, in different environments, and in different physiological states. Initially, such spectrometers could be deployed by hand in the field and linked to a GPS, but remote sensing is also possible. The data could be sent

88 Emerging Technologies to Benefit Farmers instantly to warehouses and analyzed by experts with suitable software support. The technology is not in place today, but it could revolutionize the phenotyping of plants for genetics and breeding programs and could support agricultural production. The ability to capture and transmit the images electronically would allow scientists around the world to conduct plant breeding and development studies much faster and more efficiently. Existing and evolving Tools for Transgenic Crop Improvement Biological limits on the genetic diversity of a crop’s germplasm mean that some traits cannot be readily (if at all) produced without incorporating genes from other species (referred to as transgenes) into the crop genome. Surveys of the literature show that hundreds of transgenes that affect one or more of the traits listed in Box 3-1 are known, and many of them have been field-tested. For a variety of reasons, few have been commercialized. The most common reason is that the studies have been done by academic researchers seeking to identify gene function rather than to make a com- mercial product. The most widespread commercial uses of transgenes have been to make crops resistant to insect pests (primarily lepidopterans), such as the Euro- pean corn borer and the corn root worm, and to make crops tolerant to herbicides, such as glyphosate, so that field treatments with the herbicides can take place after the crop plants have emerged from the soil. Bt Toxins Cotton that expresses a toxin protein from the bacterium Bacillus thuringiensis (Bt) is widely grown in SA and in South Africa and results in higher yields with fewer insecticide applications and fewer worker poison- ings. In China, transgenic rice crops containing Bt resistance to insects of the order Lepidoptera coupled with a variant of the (non-Bt) Xa 21 gene, which provides resistance to strains of the genus Xanthomonas, are await- ing release by the government (Huang et al., 2005). Trials of Bt cotton are proceeding successfully in Burkina Faso, and expectations are that this country maybe the first in SSA, after South Africa, to adopt a transgenic crop (ICAC, 2007) and trials are also in progress in Kenya. Bt maize for stem borer control has been commercialized in South Africa and is being field-tested in Kenya. Bt rice is now in field trials in India and China for stem borer control, and Bt eggplant is showing strong resistance to the fruit and shoot borers in trials in India, replacing up to 80 sprays per year for control. Bt genes also hold potential for control of the pod borers that attack legumes such as chickpea, pigeon pea, and cowpea.

Plant Improvement and Protection 89 BOX 3-5 Directed Evolution of Genes In recent years, a number of approaches have been developed that allow scientists to carry out “evolution in a test tube.” Through use of techniques that allow rapid shuffling of domains of genes or generation of random mutations in specific gene sequences at high frequency, scientists have been able to enhance the rate of catalysis or alter the specificity of enzymes or other proteins encoded by the altered genes (Kaur and Sharma, 2006; Babushok et al., 2007). Several recent examples relevant to agriculture are the directed alteration of an activase of Rubisco that resulted in enhanced photosynthesis and plant growth rate under moderate heat stress (Kurek et al., 2007), and the directed optimization of a bacte- rial N-acetyltransferase that, when transferred to plants, conferred resistance to the herbicide glyphosate (Siehl et al., 2007). Use of such technologies might also lead to broadening of the effectiveness or altering the specificity of Bt and other toxins important for control of insects. The Rockefeller Foundation and the U.S. Agency for International Develop- ment have been supporting projects in SSA to test the efficacy of such genes for control in SSA of lepidopteran and non-lepidopteran pests, such as pod borer in cowpea, weevils in sweet potato, and burrowing nematodes in ba- nana. There are a wider range of Bt toxins available against lepidopteran pests compared to those for the coleopteran and other non-lepidopteran pests, but there may be opportunities to use directed gene evolution (see Box 3-5) to generate new variants with altered toxicity profiles. Herbicide Resistance Transgenic crops resistant to herbicides have been widely adopted in North America and South America, especially soybean, cotton, maize, and oilseed rape. In South Africa and India, some herbicide-resistant cotton has now been released as a trait stacked with Bt. Box 3-6 describes possible opportunities for controlling weeds by engineering other forms of herbicide resistance into crop plants. Transgenes in Metabolic Pathways Although genes for insect and herbicide resistances are the most com- mon transgenes in use today, a growing understanding of metabolic path-

90 Emerging Technologies to Benefit Farmers BOX 3-6 Opportunities to Control Weeds in SSA and SA Through Engineered Herbicide Resistance Phalaris minor, Echinochloa, and Feral Rice In India, where graminicides are no longer able to control Phalaris minor (canary grass) in wheat, and other countries that cannot control feral rice and Echinochloa in rice, the most effective alternative is transgenic herbicide-resistant wheat and rice that use resistance genes that are not normally found in Gra- mineae. Such technologies are useful in wheat without fail-safe mechanisms to contain gene flow wherever there are no weedy Triticum (wheat) or Aegilops (goat grass) species (Weissmann et al., 2005). Wherever there are related Aegilops or Triticum weedy or ruderal species, fail-safe mechanisms to prevent gene flow will be essential. Containment or mitigation systems will be needed in rice to prevent the transgenes from crossing in to feral rice (Valverde and Gressel, 2005). Striga Comparative genomics has been successfully used to develop genetic mark- ers for breeding resistance to Striga in cowpea (Timko et al., 2007) and sorghum (Ejeta, 2005). A gene from rice that confers partial Striga resistance also has been isolated (Scholes et al., 2007). When the genes from cowpea and sorghum are isolated and stacked with the rice gene, it could prove useful in engineering Striga resistance in other crops. It has been demonstrated that Orobanche (broomrape), a parasitic weed simi- lar to Striga, can be controlled when the crops are transformed with genes that confer herbicide resistance at herbicide target sites of action (Joel et al., 1995; Surov et al., 1998; Aviv et al., 2002). The herbicide must be systemic and move from the site of leaf or seed application into the parasitic plant. Transgenic herbicide resistance would also be useful in sorghum, but because of feral sorghum (shattercane), it is imperative to develop and use fail-safe mechanisms that prevent gene flow where it is expected that herbicides will be widely used in the future. ways in plants suggests that it will not be long until many new transgenically enabled traits are introduced (see Box 3-7). For example, Golden Rice 2 (a variety of Oryza sativa), contains three transgenes—one from the bacteria Erwinia uredevora, one from maize, and one from the daffodil Narcis- sus—that together form a biosynthetic pathway for producing substantial concentrations of beta-carotene (provitamin A) in the rice endosperm. Although testing is still in progress and consumer acceptance of this bright

Plant Improvement and Protection 91 BOX 3-7 Engineering Plant Pathways to Decrease Postharvest Losses and Degrade Mycotoxins Isolation from major markets limits small-scale farmers in many ways, one of the most critical being the large postharvest losses incurred because of delays in getting products to markets—a challenge that is even more severe in hot tropical regions. In addition to the use of better technologies for storage and transport, plants can be bred to show reduced senescence or delayed ripening. We now recognize that senescence and ripening of fruits and vegetables involve processes akin to programmed cell death (PCD). Genes that are involved in control of PCD or of ethylene production, which also promotes ripening, will therefore need to be further tested for control of postharvest losses. A number of recent patents suggest that private enterprises are investigat- ing the possibility of producing transgenic plants that degrade mycotoxins. An amino-oxidase active against fumonisins has been isolated and cloned from black yeast, and its activity has been enhanced with mutagenesis (Duvick, 2001) and gene shuffling (Zhao et al., 2004). Human genes that express glutathione transferase, aldehyde reductase, and epoxide hydrolase, which degrade aflatoxin, have also been cloned (Bandman et al., 2003, McGlynn et al., 1995). They could be engineered into plants in different combinations to determine which, if any, have practical utility for suppressing toxin production. Such research may well elucidate the importance of the toxins in fungal pathogenicity. yellow rice remains in question, the rice offers a health benefit if eaten regularly in sufficient quantities (Enserink, 2008). A similar strategy is now being tested for development of golden cassava, sorghum, and banana that might also be enhanced for other nutritional traits such as elevated levels of zinc or iron or enhanced digestibility. Knowledge of the pathways for syn- thesis of oils in seeds is also leading to new crops with more beneficial oils, both for nutritional purposes and for use in biofuels (Dyer et al., 2008). The transferability of disease resistance genes is also of interest for crops like bananas, which are difficult to reproduce sexually. Transgenic varieties containing a resistance gene to Xanthomonas wilt from sweet pep- per are in trials in Uganda (Tripathi et al., 2006, 2008). Plant-Based Gene Silencing One of the most exciting developments in plant biology in recent years has been the discovery of small RNA molecules that play key roles in plant development and resistance to stresses. The discovery has led researchers to

92 Emerging Technologies to Benefit Farmers create vectors containing genes that encode for small RNAs that target and down-regulate or interfere with critical processes governing plant develop- ment, metabolic pathways, and also processes related to the interactions of plants with plant pests. It is probably the case that genes designed to func- tion by making RNAs with sequences that are antisense to specific target genes function via the small RNA pathways and processes to down-regulate (or “silence”) natural messenger RNAs. Although promising, the develop- ment of RNA interference (RNAi) technology is still in a very preliminary stage, especially for application in large-scale agriculture where consistency of action is essential. However, some current research strongly suggests that plant-mediated delivery of small RNAs can be used to control fungi, para- sitic plants, nematodes, and possibly many insects, such as aphids, white fly, and the bollworm or boll weevil (see Box 3-8). Site-Specific Gene Insertion Systems Homologous Transformation A long-standing goal of plant scientists and breeders is to be able to exchange alleles precisely in a directed way, which is almost impossible with standard breeding approaches. The ability to replace one allele with a more favorable one at a specific site would have a great impact on plant improve- ment and make it possible to study the functions of specific genes. Directed targeting to known sites that minimize deleterious effects and optimize gene stability and levels of expression can also greatly facilitate the more rapid movement of transgenic plants through regulatory systems. It is possible to replace an existing allele with an orthologous trans- gene in the laboratory, but this has not been efficiently reduced to practice in crops, because our understanding of the natural processes of DNA re- pair that plants use to replace and recombine alleles is insufficient. Those processes cause the transgenes to be inserted into chromosomes seem- ingly at random. Thus, breeders are usually obliged to screen hundreds of transgenic plants to find the optimal insertion event because the random locations of insertions result in large variations in expression. It would be desirable to identify the optimal insertion sites and target gene inser- tion there every time in the process of homologous recombination. Recent experiments and the development of novel systems to achieve homolo- gous recombination suggest that this goal is within reach for crops. Ho- mologous recombination can be used to insert genes, discover genes, and disrupt existing genes (Wright et al., 2005; Kumar et al., 2006; Hu et al., 2007; Lyznik et al., 2007; D’Halliun et al., 2008).

Plant Improvement and Protection 93 Zinc Finger Nucleases and Meganucleases Efficient homologous recombination relies on the existence of a double strand break in the chromosome. Thus, the challenge has been to learn how to create double strand breaks at the desired sites of insertion. Zinc finger nucleases (ZFNs) and meganucleases are tools that have been designed to achieve that. A ZFN consists of a DNA-binding zinc finger domain cova- lently linked to the nonspecific DNA-cleavage domain of a restriction endo- nuclease. A ZFN binds to a specific DNA site, and the nuclease catalyzes a double strand break. ZFNs have been shown to facilitate site-specific gene replacement in plants, but the major challenge is to learn how to make and select ZFNs that are specific for any gene that the geneticist or breeder wishes to replace. A “zinc finger consortium” has taken on the challenge, and publica- tions describing standardized reagents and protocols for engineering ZFNs by modular assembly have appeared (Wright et al., 2005). In addition, meganucleases that cleave at different sites have been isolated from differ- ent organisms (Fajardo-Sanchez et al., 2008). One company, Cellectis S.A., has also developed a high-throughput screening platform to produce a large number of different meganucleases. The further development of this technology will bring a new and powerful genetic approach to plant breeding. In addition to inserting genes precisely, it will enable promoters to be exchanged, and this will make it possible to directly alter the expression of genes. One can envision many novel applications (see Box 3-9 and Box 3-10). Recombination Systems In addition to zinc fingers, transgenes can be integrated into chromo- somes at particular locations by site-specific recombination systems (Lyznik et al., 2007). These systems rely on proteins that specialize in recombining two identical specific sequences. That enables, for example, multiple novel genes to be inserted at a target site. The so-called Cre/lox recombination system derived from bacteriophage P1 has been used for site-specific in- tegration of DNA into tobacco and rice (Day et al., 2000) and has been successfully used in wheat and rice to target single-copy insertions into lox sites placed in the genome (Srivastava et al., 2004). The lox target (a small 34-base pair DNA) is inserted into a chromosome at random. The Cre recombinase then inserts the desired transgene into this genomic target. Another system, FLp/frt, involves the flippase recombinase derived from yeast. Flp recognizes a pair of frt target sequences that flank a genomic re- gion of interest. The flp recombinase system has been used in maize (Lyznik et al., 2003) and rice (Hu et al., 2007), as have the lambda and PHIC31 integrases (Suttie, et al., 2008) and (Ow et al., 2004), but there is concern

94 Emerging Technologies to Benefit Farmers BOX 3-8 Opportunities to Apply RNAi to Agricultural Constraints in SSA and SA Control of RNA and ssDNA Viruses RNA viruses—including cassava brown streak, cucumber mosaic virus (CMV), and single-stranded DNA (ssDNA) viruses, such as African CMV in SSA and cotton leaf curl in SA—cause major losses to crops grown by small-scale farmers. Because there seems to be no strong resistance to some of the viruses available in germplasm of conventional breeding programs, the most promising strategies for addressing the pests involve the introduction of transgenes into the host plants. Strategies to overcome the RNA viruses can build on the basis of an effective trans- genic approach used to control ring-spot virus in papaya—a success story that virtu- ally resurrected the growing of papaya in Hawaii (Gonsalves, 2006). That approach, first demonstrated in tobacco against tobacco mosaic virus, involves engineering the host plant to overexpress the relevant virus coat protein gene. It now appears to be effective for many such viruses (Beachy, 1999). An alternative approach that has been shown to be useful in the laboratory uses RNAi constructs to silence key RNA viral genes (Vanderschuren et al., 2007). But when multiple viruses infect a plant, a strategy is needed to prevent one virus from producing genes that suppress the RNAi directed at silencing another. The solution might lie in the recent design of small RNAs that target viral-suppressor sequences (Niu et al., 2006). More research is needed on the mode of action of all types of suppressors of silencing. For the ssDNA viruses, RNAi that targets genes that encode a protein required for viral replication, called rep, has proved to be partially effective and is being tested against CMV (Vanderschuren et al., 2007), but the possibility remains that viral-sup- pressor genes may overcome the effects of the RNAi. Control of Other Pathogens, Pests, or Parasitic Weeds The promise of RNAi technology was recently extended significantly by the demon- stration of its use to control the bollworm in cotton. In this case, plant-mediated RNAi was used to silence a bollworm P450 monooxygenase that resulted in lowered toler- ance of the bollworm to the toxic gossypol produced by the transgenic cotton (Mao et al., 2007). Small RNAs can pass from crops to parasites, as is well-documented on the basis of technology used to control root-infesting nematodes (Huang et al., 2006). At least three groups are testing RNAi molecules that are targeted at Striga genes, or parts of Striga genes, that have no sequence homology with crop genes (de Framond et al., 2007). So far, no major success has been reported, but this new

Plant Improvement and Protection 95 research warrants attention. The knowledge base of fungal genomes is growing rapidly, and the information offers opportunities for creating mechanisms to enhance aflatoxin resistance. One possibility is to interfere directly with the metabolic pathways that lead to mycotoxin production, inasmuch as the responsible genes have been elucidated (Yu et al., 2004; Wen et al., 2005). Gressel (2008) has suggested using the information to generate RNAi constructs to inactivate one or more of the mycotoxin biosynthesis genes; these could be delivered via viral pathogens of the fungi or by expression in the plant genome. Reducing Apigenin in Fonio and Pearl Millet with RNAi The high consumption of fonio and pearl millet is associated with goiter. At its worst, endemic goiter leads to endemic cretinism, a severe form of mental retardation. In an interior area of Guinea, 70 percent of the inhabitants had goiter (Konde et al., 1994). Goiter was also found in one-fifth of children tested in the southern Blue Nile region of Sudan, an iodine-sufficient area, and the syndrome was correlated with the consumption of pearl millet (Elnour et al., 2000). Livestock eating pearl millet also suffer from hypothyroidism (Gadir and Adam, 2000). The culprit in fonio was discovered to be the flavonoid apigenin (Sartelet et al., 1996), and in pearl millet its glycoside, vitexin (Gaitan et al., 1995). Both are potent inhibitors, at low concentrations, of thyroid peroxidase, a key enzyme controlling thyroid hormone biosynthesis. If the specific flavonone synthase genes of fonio and pearl millet responsible for apigenin biosynthesis can be cloned, it might be possible to reduce apigenin in fonio and pearl millet through RNAi technology. Orthologs of those genes from Apiaceae have already been cloned, so if there is sequence similarity, it may be simple to isolate the gene from the Gramineae species involved in goiterism (Martens et al., 2003; Gebhardt et al., 2005). An RNAi construct might be made with a seed-specific promoter, turning off apigenin production only in the grain so that consumers of the grain would be protected while the stalks would retain apigenin, conserving its (probable) role as a deterrent of insect pests of the crop. On the basis all of these promising results, further exploration of the possibility of use of RNAi for control of a wide variety of important plant traits is certainly war- ranted. The potential to control critical pests like whitefly, aphids, or weevils could also be explored. In addition, necessary fundamental research to better understand how RNAi might be used in crop plants includes studies to determine how and where movement of small RNAs occurs, whether plants discriminate between different small RNA molecules that move between the plant and the pest, and what size of small RNA molecules can move. We also need to know whether a target organism can evolve resistance to RNAi and, if so, at what rate.

96 Emerging Technologies to Benefit Farmers BOX 3-9 Disrupting Plant-Virus Replication The ssDNA viruses, particularly geminiviruses, have high rates of mutation and recombination that make it difficult to target specific viral sequences (Legg et al., 2006; Arguello-Astorga et al., 2007). New technologies might have a role to play in controlling these deadly, highly variant viruses, such as the African cassava mo- saic virus that is causing the current pandemic in cassava (Mansoor et al., 2003, 2006; Legg and Fauquet, 2004; Vanderschuren et al., 2007). For viruses in the Mastrevirus genus, such as the one that causes maize streak, maize engineered to overexpress a transdominant mutant rep protein holds a great deal of promise (Shepherd et al., 2007). Three other approaches worthy of exploration for control of geminiviruses are the use of peptide aptamers that target conserved regions in rep proteins (Lopez-Ochoa et al., 2006), expression of a gene that encodes a non- specific ssDNA-binding protein (Claude Fauquet, Donald Danforth Plant Science Center, personal communication), and the use of an artificial zinc finger protein that can be designed to be inserted into and disrupt the origin of replication of the virus (Sera, 2005). Even stronger control might come from targeting the origin of replication of the deadly satellites and thus controlling the emergence of new complexes. about damage caused by the action of recombinases on cryptic excision sites in the genome. An approach previously applied to animals was adopted by Agrisoma Biosciences, using sequences homologous to ribosomal DNA in the trans- formation vector, alongside the genes of interest. After insertion of the vector into plant cells, scientists stimulated recombination of the novel genes into the host ribosomal DNA, where the genes reside in an amplified structure in the regenerated plants. Additional genes can be added at the same general sites by similar homologous recombination events. Those approaches facilitate the stacking of new traits at valuable loci in a modular fashion and can integrate new genes at a site in the genome that has already been found to support strong constitutive expression, avoiding disruption of existing genes and adverse agricultural effects. If brought into routine use, the methods will reduce the amount of work needed to stack multiple transgenes or to introgress them between lines in a breeding pro- gram and may ease regulatory approval since there is often a requirement in many jurisdictions to provide the DNA sequences flanking the insertion sites, which will be the same whenever site-specific integration is used.

Plant Improvement and Protection 97 BOX 3-10 Potential Transgenic Approaches to Protect Sorghum Against Birds Birds have been chemically controlled with an organophosphate insecticide, fenthion, applied with back-pack sprayers (Mundy, 2000; van der Walt, 2000), but there are better biotechnology alternatives. A white, non-tannin containing sorghum variety, Ark-3048, is not attacked by birds (York and Daniel, 1991). Its seeds are high in dhurrin, a natural cyanogenic glycoside often found in seedlings and stalks of sorghum (Kahn et al., 1997). The cyanogenic compound dissipates by maturity, when the birds no longer find the seeds palatable (Alkire, 1996). Breeding with Ark-3048 has not advanced, because derived progenies have yield penalties (Gebisa Ejeta, Purdue University, personal communication, December 11, 2006). It might be possible to generate a sorghum that makes dhurrin only in the developing seeds, so that livestock that eat sorghum forage will not be poisoned. There might be less yield drag if the dhurrin genes were put under a high expression seed-specific promoter and not expressed in other tissues. The relatedness of sorghum species and the existence of weedy feral forms of the crop (Ejeta, 2005) present a gene-flow hazard. A morphological solution would be to put the grains closer together on the stalk, make them harder to peck apart, and ensheath the grain heads to make them less conspicuous. The Mexican Amerindians did that in maize without genetic engineering by selecting genes that turned teosinte, with its open head, into maize, with its less accessible cob (Doe- bley, 2004). The open inflorescences of sorghum are reiteratively branched like maize ramosa mutants (Vollbrecht et al., 2005). In sorghum, delayed production of spikelet pairs correlates with a protracted onset of ra1 expression (Vollbrecht et al., 2005). One might ask what would appear on a sorghum plant if the native ramosa were transgenically replaced with the maize genes; the answer would require extensive genomic and biological research, which might yield a bird-proof sorghum. Meiotic Recombination During meiosis in plants, the homologous chromosomes of egg-forming and pollen-forming cells undergo recombination in a process that results in new combinations of genes (alleles). A high rate of recombination at many places in chromosomes leads to a greater number of new gene combina- tions. A low rate of recombination maintains existing gene combinations. If the rate of recombination (Kitada and Omura, 1984) and the positions of recombination along the chromosomes could be controlled and directed

98 Emerging Technologies to Benefit Farmers to create desirable recombinant genomes, plant breeding might be more efficient. It has already been learned by comparing recombination maps with physical maps that recombination occurs more often near the ends of chromosomes (See et al., 2006). Recombination involves cleavage of DNA molecules and repair processes that can lead to resynthesis of the par- ent strand or to a crossover and a recombinant chromosome (Wijeratne and Ma, 2007). The multistep processes are complex and involve many proteins. Before processes can take place at the level of DNA, specific polynucleotides in the DNA molecules have to be accessible to the protein complexes and to each other. It now appears that one level of control of recombination involves the regulation of chromatin condensation and the coordination of the condensation between different chromosomal regions. That is the basis of variation at the Ph1 locus, which affects recombination between simi- lar chromosomes in wheat (Griffiths et al., 2006). The genetic control of chromosomal condensation into heterochromatin is now known to involve small RNAs and epigenetic changes in histones and DNA. As research proceeds along this path, it may become possible to fathom how to locally control the degree of chromatin condensation during meiosis and so direct the places and frequency of recombination during meiosis. It will not be easy to implement in a single crop or to apply to all crops, so this should be seen as a long-term opportunity. The Ph1 gene that regulates recombination frequency between chro- mosomal homologs in wheat is useful because recombination between the more distantly related homologous chromosomes occurs when it is absent (Griffiths et al., 2006; Wijeratne and Ma, 2007). This allows additional alleles to be brought into wheat from wild relatives of cultivated wheat. This illustrates another potential use of manipulating recombination: the incorporation of new genetic material into crops via wide hybrids. That use has had little success for many reasons, including its bringing many unwanted and deleterious genes in addition to those desired, but it should be revisited when new understanding and tools are available. As discussed earlier, it may be possible to insert into a chromosome sequences that pref- erentially undergo recombination at specific locations that is catalyzed by specific nucleases and in this way direct meiotic recombination events to some positions and away from others. Artificial Chromosomes Crop improvement involves combining the best alleles for key genes in a single variety. In a transgenic approach, that is accomplished by the stacking of various genes, preferably at a single locus so that the introduced genes do not segregate from each other in later generations. Homologous

Plant Improvement and Protection 99 recombination and site-specific integration are beginning to offer breed- ers the potential to bring multiple genes together at a single site, making it easier to control their expression or to delete them selectively, but an- other novel approach has recently been pioneered by such companies as Chromatin. Chromatin has developed a method of synthesizing a mini-chromosome by linking genes of interest to a large piece of maize DNA that encodes satellites, retroelements, and other repeats commonly found in maize cen- tromeres. Other groups have developed artificial mini-chromosomes using telomeres (Lamb et al., 2007; Yu et al., 2007; Birchler et al., 2008). Those elements of DNA confer on a chromosome the ability to be divided regu- larly between daughter cells at mitosis and meiosis. When such artificial chromosomes were introduced into maize cells by particle bombardment, the new chromosomes were shown to be regularly inherited in plants re- generated from the cells (Carlson et al., 2007). The technology is new, and there are technical issues to be satisfied, including the stability and fidelity of gene expression and the reliability of inheritance over the many generations associated with agricultural seed production. Other concerns are the stability, rearrangement, and expansion or contraction of the repetitive sequences in the centromere regions and the possibility of epigenetic silencing of gene expression over generations and in other genetic backgrounds (Dawe and Henikoff, 2006; Talbert and Henikoff, 2006; Carlson, 2007). Species-specific systems for the major crops of SSA and SA would need to be developed to use the technology. As the availability of valuable genes for crop improvement increases, it will be necessary to address questions of where and how to insert multiple genes for long-term utility. Given the po- tential power of this technology, a large number of projects using artificial chromosomes could be envisioned. Nitrogen fixation (discussed in greater detail in Chapter 5) would be one such project. Conceivably, it would be possible to develop transgenic, nitrogen-fixing crops—such as rice, wheat, and maize—by adding an artificial chromosome with 20 or so genes known to play a role in fixing nitrogen. There is debate, however, in the scientific community about the metabolic and yield costs to the plant that would occur by adding this trait, so a project of this nature would be considered highly experimental. Apomixis Hybrid seed is more expensive to produce than certified seed and must be purchased every season because of segregation of properties in the off- spring of the hybrids. Farmers who cannot afford to buy hybrid seed every season and opt instead to plant seed saved from a previous harvest forgo the benefits of heterosis—the vigorous performance of hybrid seeds—that

100 Emerging Technologies to Benefit Farmers include higher yields and greater resistance to pests and diseases. If it were possible to maintain the hybrid genotype in seed from one generation to the next, those benefits also would be preserved, so certified weed-free and pathogen-free seed could be produced at a much lower cost or farmers could save seed from one year to the next. In many wild plant species, the perpetuation of the hybrid genotype is accomplished through apomixis, a process in which progeny seed pro- duced in a plant without the sexual fertilization of cells give rise to embryos (Koltunow and Grossniklaus, 2003). The asexual event leads to the propaga- tion of hybrid genotypes in the following generations. The genetic basis of the different forms of apomixis in wild plants is complex, but it is conceivable to harness this mechanism as a technology (Grimanelli et al., 2001). Research to understand the process is proceeding (Grimanelli et al., 2001; Catanach et al., 2006). There is evidence that one or two dominant genes are involved in some systems with a large chromosomal segment that does not recombine. With additional research, it might be possible to design transgenes and insert them into crop plants to change the mode of plant seed production from sexual fertilization to apomixis. Whether it is possible to find genes that provide such a switch and to deploy them while main- taining high seed yields is an open question. Many believe it sufficiently important to continue research toward that goal. Alternatives to Bt Controlling insects that feed on crops can reduce not only losses from pest damage but the incidence of disease. For example, stem borers are vectors of Fusarium spp., and grain weevils, especially the lepidopteran ear borers, carry Aspergillus spp. Those two genera of fungi are responsible for the production of mycotoxins. Crops that have been genetically engineered to produce Bt insecticidal proteins experience less insect damage. The effect of Bt is greatest in seasons when fumonisin concentrations are the highest because of heavy infestations with stem borers (Munkvold et al., 1999; Munkvold, 2003; de la Campa et al., 2005). To further reduce mycotoxins, plants will need to have stacks of multiple genes that encode activities that kill the fungi and the coleopteran and lepidopteran insects that are vectors of the fungi in the field and in storage. It might be even better if genes are stacked that encode enzymes that degrade the mycotoxins. Toxins from other pathogens carried by insects are a potential source of novel insecticidal compounds. Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes that are lethal to a wide array of insects and were effective when expressed in Arabidopsis (Liu et al., 2003). Other major foci for research could include genes from plants (such as genes that produce enzyme inhibitors and lectins) and animals, including insects (such as genes that produce biotin-binding proteins, neurohormones, venoms,

Plant Improvement and Protection 101 and enzyme inhibitors). Fungi have been underexploited, particularly with respect to pathogens carried by insects, even though they are exceptionally rich sources of novel biologically active substances (Isaka et al., 2005). The nearly 1 million arachnid toxins could probably be a major re- source for genetically modified plants and biopesticide delivery systems (Edwards and Gatehouse, 2007; Whetstone and Hammock, 2007). They include toxins specific for many organisms, including microorganisms, and their potential appears virtually limitless if they can be provided with suit- able delivery systems. As the history of insect control teaches, resistance to almost all interventions evolves eventually. Thus, although the effectiveness and specificity of Bt toxins in transgenic plants are extremely important for the future (Federici, 2007; Uneke, 2007), a multitude of stacked genes will make it harder for insects to evolve resistance. Sentinels of Drought, Disease, and Deficiencies Skilled farmers can readily recognize deficiencies in their crops, but it is often too late to supply a remedy and retain the yield, because the crops have already reorganized their internal chemistry to cope with the stress and consequently have suppressed growth or initiated senescence or irre- versible death pathways. It is, however, possible to detect internal shifts in chemistry long before signs of the stress appear. The question is how to get such information to the farmer. Can the exquisitely sensitive and specific molecular sensing mechanisms of plants be harnessed to tell farmers the conditions of their fields, their soils, their ecologies, and their crops daily and at low cost? Research with Arabidopsis has shown that it is possible to design transgenes and insert them into plants so that the plants serve as sentinels or reporters that can be observed by the farmer and indicate, for example, deficiencies in soil or water levels or an early stage of disease (Jefferson, 1993; Liew et al., 2008; Mazarei et al., 2008). Such genes consist of a pro- moter that responds specifically to the deficiency or stress of interest and is connected to a reporter that stimulates production of an easily visible product, for example, a pigment. The color or amount of pigment would help the farmer to decide how to best use available resources and when to take precautionary action to protect the crop. Signals could help a farmer to determine, for example, the most and least productive areas of land and which nutrients should be added to the soil and how much. That could prevent the wasteful use of nutrients, which is uneconomical and polluting. Early signs of water stress would be revealed in the field, and water could be applied to parts of the field that need it. The reporter plants could be non-crop plants that do not interbreed with the crop but are physiologi- cally matched to the crop. Ideally, they would be scattered across the field in strategic places and not harvested with the crop.

102 Emerging Technologies to Benefit Farmers Sentinels could also be useful to plant breeders, who face a major prob- lem in simultaneously comparing different genotypes for many characters. Use of sentinels in breeding materials would, for example, make it easier to know whether one plant used water or nitrogen more efficiently than another. Reporter genes could be left in the commercial variety or crossed out later in the breeding program. Chemical-Induced Switching A forward-looking type of crop is one with qualities that can be modi- fied on the basis of weather, market conditions, or local need. One can imagine that sorghum, for example, could be grown for its seed, as now, or for its biomass for energy purposes. Specialized crops can exist for the two kinds of applications, but what if farmers could stop flowering and seed production, and instead, produce more vegetative biomass, in light of ac- curate market or weather predictions that would favor biomass? A farmer might seek to turn a carbohydrate crop into a protein-enriched crop because of failure to obtain protein from other sources, or it might be desirable to accelerate flowering to meet an off-season demand or to enable a different crop rotation. Many such scenarios can be envisaged for SSA and SA. Today’s science is producing knowledge that is making such possibili- ties more realistic. Genes that can stop flowering, bring flowering on earlier, and control major pathways leading to different end products are being found. Breeders will learn how to manipulate them to the extent that use- ful genetic variation is available. However, transgenes that can create such shifts in traits can be placed under the control of promoters that respond to specific chemicals, and these chemicals would be applied by a farmer to initiate the changes (Box 3-11). For such transgenic crops to become practical, it would be essential to discuss their possible value with a farmer and then to seek promoters that can respond specifically to easily obtained chemicals that can be sprayed on crops without damaging people or the environment. Such chemicals exist, and it has been shown that promoters that respond to them can be found (Girke et al., 2005). In general, the public sector could benefit from having available a much broader suite of tissue-specific or inducible promoters than is available today. Current Bottlenecks in crop improvement Transformation and Regeneration The ability to introduce new genes into plants depends on many fac- tors, especially the frequency with which transformed cells can be induced to divide, form embryos, and then form plantlets. The genetics of the host

Plant Improvement and Protection 103 BOX 3-11 An Inducible Suicide Gene for Weed Control? The crop-parasitic weed Striga hermonthica is an obligately outcrossing spe- cies and must be cross-pollinated. If this species were transformed with a multi- copy transposon bearing a lethal (suicide) gene under the control of an inducible promoter, a small number of such plants could be introduced into fields. With a dominant gene, only half the progeny from crosses would bear the transgene; because of the nature of a multicopy transposon, virtually all progeny of all generations would bear the potentially lethal transposon. After about five gen- erations, when the whole population bears the gene the inducible transgene can be turned on, killing the Striga (Gressel and Levy, 2000). The concept of using multicopy transposons bearing inducible lethal genes was first elaborated for in- sects (Grigliatti et al., 2001), but it could be applied to any obligately outcrossing weed (Gressel, 2002). Two kinds of inducers would be ideal for this situation: a chemical inducer emanating from a crop root and a radio-wave-inducible system, which is still in the realm of science fiction. The required genetic systems might come from bats, which perceive radio waves in their echolocation systems. plant are influential; many elite cultivars are among the most difficult to grow in tissue culture and to induce to form new embryos. Being able to easily regenerate crops from tissue culture would speed the process of plant improvement (Gelvin, 2003; Shrawat and Lorz, 2006). It is now possible to add cell division-promoting genes to the transformation vectors used to introduce genes into a plant, and there is evidence that this can substantially boost regeneration efficiency. For example, scientists at Pioneer took a repA gene from wheat dwarf virus and put it into maize. The gene stimulates the cell cycle and results in many rapidly growing colonies per embryo; the effect is greater when the gene is under the control of a more active promoter. The method was able to substantially increase the number of maize transgenic lines (Gordon- Kamm et al., 2002). The Lec1 gene from Arabidopsis produces a similar effect (Lowe et al., 2002). Some plants, e.g., cowpea, regenerate with reasonably high frequency but only a low proportion of such can be transformed, and the challenge is to facilitate transformation in regenerable tissues. In this regard, worthy of further investigation is a recent report that the use of an anti-apoptosis gene from animals prevented death of banana cells transformed with Agro- bacterium and led to a large increase in transformation efficiency (Khanna et al., 2007).

104 Emerging Technologies to Benefit Farmers If such technologies were deployed in the germplasm of SSA and SA crops, the production of transgenic crops would much more efficient. That would enable scientists and breeders to make and evaluate many more transgenic plants from which to select optimum forms with the probability of better products as a consequence. The advances could be beneficial in early research phases and in product development. For regulatory and other reasons, genes that stimulate regeneration might have to be deleted from commercial crops. That can be done by placing the regeneration-stimulating genes and the desired trait genes on separate vectors, selecting elite progeny, and then deleting the regeneration-stimulating genes by screening progeny with only desired genes in later generations; or the regeneration genes might be expressed only transiently during the early stages of transformation and selection. Shortcomings of Transgenics The many issues and problems associated with the science and com- mercial deployment of transgenes include the following: • Variable expression and instability over generations that requires screening many events to obtain both stability and required level of expression. • Cost of regulation and the additional time required for transgenic processes. • Silencing of transgene expression by other genetic elements in the plant. • Regulatory demands to remove selectable markers associated with transgenes. • Inefficient transformation processes for certain genotypes. • Consumer and political acceptance, even when improvements are valuable. • Outcrossing to nontransgenic relatives. • Intellectual-property and freedom-to-operate issues. • Costs, if crops have to be kept separate from nontransgenic versions. In spite of all those challenges, it would be short-sighted to continue trying to solve crop production constraints and breeding inefficiencies decade after decade without using transgenes. A strategic plan is needed to incorporate transgenes into SSA and SA crops for the sole purpose of relieving or removing the constraints that contribute to poverty. Because the plant science community has made a major investment in the use of trans-

Plant Improvement and Protection 105 genes to evaluate gene-trait associations, there will continue to be a large supply of potentially useful transgenes worthy to consider for applications in SSA and SA crops. Moreover, many of the challenges listed above are slowly being ad- dressed. The Public Intellectual Property Resource for Agriculture (PIPRA) is an example of an effort actively engaged in enhancing the freedom-to- operate (to use patented genes) in specialty crops and crops developed for humanitarian purposes. Some of the scientific approaches discussed in this chapter could help to speed the regulatory process. For example, the ability to integrate genes in specific, pre-determined, optimized sites in the genome should reduce significantly the number of events that need to be generated and tested. Site-specific gene integration might also make it more acceptable to approve a particular gene construct that could then be used for transformation of many related varieties (as opposed to event-specific regulation)—something that is critical for vegetatively-propagated crops such as cassava, banana, and sweet potato where back-crossing a transgene into other varieties is not feasible. Studies on gene silencing are also leading to the design of constructs that avoid problems with silencing of transgenes. Finally, as discussed in the next section, technologies are emerging to elimi- nate gene flow and the problem of outcrossing with other plants. Control of Gene Flow from Transgenic Plants Fail-safe mechanisms are needed to contain gene flow and mitigate the effects of a transgene’s escape to wild and weedy relatives of the crop (Valverde, 2005). Various containment procedures have been proposed, but all seem to be unidirectional or leaky, and none has been tested in the field (e.g., see Daniell et al., 2002). For crops which are sterile—the classic one being banana—gene flow is simply not an issue. For other cases where the flower or seed does not add significant value to the product, such as cas- sava or some trees, techniques are available to repress flowering and seed production through down-regulation of key flowering genes. Another ap- proach is to introduce the transgene into the chloroplast DNA so that the pollen will not carry the gene (Daniell et al., 2002). While this can work in some cases, there are cases where genes may be transmitted through the pollen, but the relative of the crop can be the recurrent pollen parent, allowing gene transfer albeit more slowly. Other approaches may involve fruit-specific excision of transgenes or development of strategies in which the fitness of the resulting hybrid is compromised. These latter technologies have been suggested as a means to control feral rice (Valverde and Gressel, 2005); this idea has been validated with tobacco (Al-Ahmad et al., 2004) and oilseed rape (Al-Ahmad et al., 2006) but not yet with rice. Examples of

106 Emerging Technologies to Benefit Farmers mitigation genes that could be used to render hybrids between rice and feral rice noncompetitive include those responsible for dwarfism, non-shatter of seed, and lack of secondary dormancy (Gressel, 2008). Understanding DNA Satellites Associated with Geminiviruses One of the greatest challenges in controlling ssDNA viruses, such as the geminiviruses, is the emergence of DNA satellites associated with the viruses that, in some cases at least, are believed to serve as extremely ef- fective suppressors of silencing and thus enhance disease symptoms. The emergence of two such satellites is responsible for the breakdown of crop resistance of cotton in cultivars that were developed to be resistant to cotton leaf curl virus in Pakistan (Amin et al., 2006; Briddon and Stanley, 2006; Mansoor et al., 2006). The existence of DNA satellites in cassava may help to explain the rising pandemic in SSA of cassava mosaic disease (Ndunguru, 2005). An intensive research effort to determine the fundamental nature of those DNA satellites is needed so that the products they encode and how they overcome resistance alleles in the target crops can be understood. The research should be coupled with strong support for development of regional diagnostics to develop baseline information and carry out surveys that can follow the emergence of new disease threats, especially emerging satellite DNAs—information that will need to be communicated rapidly to breeders so that they can test all their resistance alleles for effectiveness in the pres- ence of newly emerging viral sequences. Plant Protection with Classical and Genetically Engineered Biocontrol Agents The previous sections of the report focused on manipulating the plant to introduce improved traits, including resistance to diseases and pests. This section explores the potential for other organisms to be used to protect plants from insects, weeds, and other pests. Biological Control of Insects Use of Natural Predators Insect biological control (biocontrol) consists of the release of specific natural enemies, usually from the place of origin of an exotic pest. Parasit- oids are the most frequently used natural enemy group for biocontrol. The long history of plant breeding as the focus of plant protection efforts in Africa (Hahn et al., 1989) has meant relatively weak support for biocontrol initiatives. However, most food in Africa is produced from exotic crops that

Plant Improvement and Protection 107 originated in South America, southeastern Asia, or SA. With foreign insect species invading Africa at an increasing rate and threatening agriculture and conservation, biocontrol is increasing relevant. Although biocontrol is not a quick fix and is usually doomed to failure if there has been no preliminary research, Africa has seen some of the most successful examples of classical biocontrol, in part because the introduced agents have not been exposed to pesticides, given the low application rates common in subsistence farming. The data in Table 3-1 demonstrate the effects of three biocontrol technologies. In terms of yield, they offer ben- efits comparable with those of long-term breeding programs for maize and cassava. The use of biocontrol agents can help to preserve the biodiversity of crop varieties inasmuch as the agents can be used with all varieties whereas conventional or transgenic breeding of varieties with insect-resistance traits usually involves a small number of varieties. Some possibilities are described below. Control of Whitefly.  The world’s most destructive whitefly pest, Be- mesia tabaci, is a target worthy for biocontrol. Its genetic diversity, its wide range of host crops and other plant species that lead to its ability to transmit more than 70 disease-causing viruses, and its environmental adaptability make this pest particularly challenging, and control will require multicom- ponent, integrated pest management (Legg and Fauquet, 2004; Legg et al., 2004). Cassava, an important crop in Africa, is being severely affected by TABLE 3-1  Economic Impact Analysis of Current Biocontrol Projects in Africa Pest Species and Control Agent Time of First Losses and Time of Start Reduction Occurrence in Yield of Campaign Area in Loss Cassava mealybug, 40% Encyrtid wasp, 27 African nations 90-95% 1973 1981 Cassava green mite, 35% Phytoseiid mite, Nigeria, Ghana, Benin 80-95% 1971 1983 Mango mealybug, 90% Encyrtid wasp, Benin 90% 1980s 1987 NOTES: Estimated savings from the biocontrol projects in millions of U.S. dollars were 7,971- 20,226 (cassava mealybug), 2,157 (cassava green mite), and 531 (mango mealybug). Estimated saving were achieved for costs far below 1% of research costs. SOURCE: Neuenschwander, 2004. Reprinted by permission from Macmillan Publishers Ltd: Nature (Neuenschwander, 2004), © 2004.

108 Emerging Technologies to Benefit Farmers the spread of two virus groups by B. tabaci: CMV disease, caused by gemi- niviruses, and cassava brown streak, caused by an ipomovirus. Little has been done to address the whitefly situation. Control of the highly fecund B biotype has been achieved in the United States and else- where through a process of conserving and augmenting natural enemies and introducing new ones from other locations with similar environments (Goolsby et al., 2000; Hoelmer et al., 2000). Although the B biotype is not the form of the pest that is causing problems in the main tropical zones of SSA, the strategy used to control it elsewhere provides a model for a potential biocontrol program in Africa. A concerted effort is needed to characterize the genetics and biotypes of B. tabaci in Africa, to explore interactions of the pest with natural enemies and host plants, and to test the extensive fauna of B. tabaci parasitoids worldwide to examine the pos- sibility of introducing additional parasitoid species. A smaller but important adjunct to the effort would be a program to tackle alien invasive whitefly species. One of the most destructive of these is the spiralling whitefly, Aleurodicus disperses, major infestations of which occur on a wide array of annual and perennial crops in eastern Africa. Fruit trees are an important source of income for farming communities in this coastal zone, and coconut, papaya, guava, and avocado are all seriously affected. Occurrences in both northern and southern coastal zones mean that spread is continuing, but in the absence of trained observers it is prob- ably being missed. Inasmuch as effective control has already been achieved in western Africa through the spread of parasitoids, an effective solution could be possible in a relatively short time. Control of Pests of Cowpea.  The cowpea aphid originated in the Mid- dle East and south Central Asia, where it is seldom recorded as reaching pest status because a multitude of parasitoids of Aphis craccivora, such as Trioxys spp. in Pakistan and India, control this pest in a variety of envi- ronments and on different crops (Singh and Agarwala, 1992). Considering that no parasitoids attack this pest in SSA (Soukossi, 2001), introduction of climatically adapted strains might reduce aphid numbers below the dam- age level (Manuel Tamo, IITA-Benin, personal communication). A potential biocontrol agent for cowpea bruchids is the parasitoid Dinarmus basalis (Amevoin et al., 2007). Similarly, the cowpea pod borer, Maruca vitrata, is controlled in Asia by three braconid wasps, three ichneumodids, and a tachinid fly (Huang et al., 2003). Preliminary studies suggest that among these Apanteles taragamae and Nothura maculosa (with up to 63 percent and 40 percent parasitism, respectively) are promising candidates to exploit as biocontrol agents against the pod borer in western Africa, where it has few natural enemies. Biocontrol with pesticides based on natural pathogens is one method

Plant Improvement and Protection 109 for replacing chemical insecticides (Uneke, 2007). Biocontrol of insects has relied mainly on the bacterium Bacillus thuringiensis (Bt) and the Cry (for crystal) protein (protoxin) it produces that is highly toxic to some species of insects after ingestion but safe for most nontarget organisms. The success of using whole Bt is due to the relative ease of mass-producing products based on it (Federici, 2007). However, a problem of toxin resistance is emerging in some insect species (Poopathi and Tyagi, 2006; Federici, 2007). Genetic engineering has been used to develop more potent recombinant bacteria to produce multiple toxins that are more cost-effective and less prone to resistance (Federici, 2007). Some of the major insect pests of cowpea can be controlled with inun- dative applications of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae (Tamò et al., 2003). Fungi are the most common pathogens in nature that attack thrips and aphids, and their mode of ac- tion—direct penetration of the cuticle—makes them suitable candidates for controlling these pests (St. Leger and Screen, 2001). Such fungi may also be useful for controlling storage pests, such as the bruchid beetle, which is able to cause losses of 60 to 80 percent (Glitho and Nuto, 1987). The application of B. bassiana was able to protect stored cowpea for up to 6 months (Cherry et al., 2007). Viruses also have the potential to be used as biocontrol agents for bit- ing insects. A hitherto unknown nuclear polyhedrosis virus (MaviMNPV) was found to infect the cowpea pod borer (Maruca vitrata) moth larvae in Taiwan (Lee et al., 2007) and was later exported to Africa for trials. Preliminary observations indicate that the virus can control M. vitrata, and an effort is under way to conduct surveillance of the pest with pheromone- based traps in different cowpea cropping areas. Once that is achieved, the traps could be used to derive an intervention threshold for spraying with MaviMNPV. Genetic Engineering for Biocontrol and Biopesticides An advanced approach could remedy the perceived deficiencies in natu- rally occurring biological pesticides by molecular manipulation to improve virulence (speed of kill), restrict or widen the host range or reduce inoculum loads, and alter saprophytic competence. Such technology has been used to produce hypervirulent viruses and fungi (Zlotkin, 2000; Wang and St. Leger, 2007). Classical insect biocontrol is being challenged to improve its success rate, robustness, and reliability. The use of genetics to enhance efficacy of natural enemies has attracted a lot of discussion but delivered little thus far (Poppy and Powell, 2005). It might be possible in the future to use trans- posable elements that allow insects to be engineered with a variety of traits,

110 Emerging Technologies to Benefit Farmers making genetic engineering of parasitoids likely (Atkinson et al., 2001; Grigliatti et al., 2001). Parasitoids show remarkable phenotypic plasticity because of associative learning. Understanding the genetic control of learn- ing and the ability to select parasitoids for learning ability is an exciting prospect if research on gene-environment interactions can be studied. Suicide-Inducing Genes An emerging approach to pest management is genetic modification of the insect pest to target it for biocontrol. A possible biological pest man- agement system, dubbed TAC-TICS (Grigliatti et al., 2001), proposes to transform the whitefly pest with a multicopy deleterious transposon bearing an incapacitating gene with an inducible promoter. The transformed insect would be released into the population for dissemination of the transposon; after spread, a chemical switch would turn on the incapacitating gene. Prog- ress in insect sciences makes each of those steps feasible, but the scheme would require considerable research to implement in the field. Recent suc- cesses have been achieved in transforming mosquitoes to make them un- able to transmit the malaria parasite and fitter than wild-type mosquitoes (so that they replace them) (Marrelli et al., 2007). It might be possible to produce transgenic whiteflies that carry a lethal gene under the control of a promoter that is turned on by the presence of plant pathogenic viruses so that only whiteflies carrying the viruses die. Biological Control of Weeds Insects are not the only organisms for which there are applications of biocontrol. For example, fungi have been isolated that control Striga in limited inundative biocontrol trials. There has been considerable success in applying fungal inoculum as a seed treatment, but support has not been available for testing and developing it on a large scale (Beed et al., 2007). It has been suggested that transgenic fungi with hypervirulence genes could be used to further increase efficiency, and there has been laboratory-scale success with this approach using Orobanche as a model for Striga. If the transgenic hypervirulence approach is used, genes would have to be added as a fail-safe mechanism to prevent spread and mating with other fungi (Gressel et al., 2007). Similarly, highly specific pathogenic fungi that at- tack Echinochloa have been isolated and tested in rice paddies (Zhang and Watson, 1997; Yang et al., 2000); these, too, could be genetically enhanced to increase virulence (Vurro and Gressel, 2007). Cultivating the perennial legume Desmodium between crop rows has been somewhat successful in controlling Striga. It secretes Striga-killing allelochemicals. The technology is limited to where Desmodium will grow

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Increased agricultural productivity is a major stepping stone on the path out of poverty in sub-Saharan Africa and South Asia, but farmers there face tremendous challenges improving production. Poor soil, inefficient water use, and a lack of access to plant breeding resources, nutritious animal feed, high quality seed, and fuel and electricity-combined with some of the most extreme environmental conditions on Earth-have made yields in crop and animal production far lower in these regions than world averages.

Emerging Technologies to Benefit Farmers in Sub-Saharan Africa and South Asia identifies sixty emerging technologies with the potential to significantly improve agricultural productivity in sub-Saharan Africa and South Asia. Eighteen technologies are recommended for immediate development or further exploration. Scientists from all backgrounds have an opportunity to become involved in bringing these and other technologies to fruition. The opportunities suggested in this book offer new approaches that can synergize with each other and with many other activities to transform agriculture in sub-Saharan Africa and South Asia.

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