African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 3, Num. 2, 1995, pp. 161-170

BIOTECHNOLOGY FOR SORGHUM IMPROVEMENT

J.L. BENNETZEN

Department of Biological Science, Purdue University, West Lafayette, IN 47907, USA

Code Number: CS95022
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ABSTRACT

Modern molecular technologies will promote tremendous change in nature and efficiency of agricultural production. DNA markers can be used to enhance breeding programmes now, and genetic engineering will play an ever-more-important role. Different crops, and a single crop in different environments, will vary in the nature and degree to which these technologies can or should be utilized. Sorghum's strengths, particularly its high level of productivity under adverse conditions, make it a very important target for continued improvement. Molecular techniques will allow exceptional advances in the productivity of a crop species, in proportion to the effort applied to that species. Hence, sorghum will face more direct competition with other crops (e.g. maize and rice) than was previously possible. In order to continue its major contribution to world agriculture, sorghum must utilize its relative strengths and acquire new capacities. Fortunately, many of the tools developed in other grass species can be directly applied to sorghum improvement, partly due to the high degree of conservation in grass gene content and map co- linearity. Appropriate applications of biotechnology will often be very different for different sorghum growers and users. However, with thoughtful selection and employment of pertinent biotechnology, sorghum production should be able to maintain or increase its agricultural significance.

Key Words: Breeding, DNA markers, genetic engineering

RESUME

Les technologies moleculaires modernes vont entrainer des changements importants dans la nature et l'efficience de la production agricole. Les marqueurs DNA peuvent etre utilises pour accelerer les programmes d' amelioration tandis que le genie genetique pourra jouer un role pins que jamais important. Differentes plantes, aussi bien qu'une seule plante, cultivees dans differents environnements pourraient varier dans leur nature selon le degre avec lequel ces technologies pourraient etre utilisees. Les points forts du sorgho, particulierement son haut niveau de productivite sous des conditions adverses de production font de cette culture une cible importante pour une amelioration continue. Les techniques moleculaires permettront de faire des progres exceptionnels dans l'augmentation de la productivite des especes de cultures, selon les efforts qui seront mis sur ces especes. Ainsi, le sorgho fera face a la competition des nutres cultures comme le mais et le riz. Pour maintenir sa majeure contribution a l'agriculture mondiale, le sorgho doit utiliser ses points forts en vue d'acquerir des nouvelles capacites. Heureusement que pinsieurs outils developpes pour

d'nutres graminees peuvent directement etre utilises pour l'amelioration du sorgho grace partiellement a son niveau eleve de conservation de genes et de colinearite. Les applications appropriees de biotechnologie seront souvent tres differentes pour differents producteurs ou utilisateurs de sorgho. Cependant, a l'aide d'une selection bien pensee et de l'usage pertinent de la biotechnologie, la production du sorgho pourrait maintenir et accroitre son importance agricole.

Mots Cles: Amelioration, marqueurs DNA, genie genetique

INTRODUCTION

Sorghum (Sorghum bicolor L. Moench) ranks fifth in world grain production, serving as the stuff of life for more than 300 million people in the semi-arid tropics, and is an important forage crop in dry land areas worldwide (Doggert, 1988). Despite its socio-economic importance, sorghum has received relatively little basic investigation. The limited resources applied to the study of sorghum have been concentrated on applications that could directly contribute to the continued success in improvement of sorghum by conventional breeding.

The advent of molecular technologies for the mapping and engineering of complex genomes now permits wholly new approaches to crop improvement. Sorghum has not yet received much attention in this regard, but the initial efforts to apply molecular technologies have yielded the first genetic maps of sorghum (Hulbert et al., 1990; Binelli et al., 1992; Whitkus et al., 1992; Melake-Berhan et al., 1993; Chittenden et al., 1994; Ragab et al., 1994; Pereira et al., 1994; Weerasuriya, 1995) and successful transformation (Casas et al., 1-993). These two accomplishments will now allow improvement of sorghum both by greatly-enhanced traditional breeding and by genetic engineering. The chief limitations in undertaking this work today are not technical; primarily, we need more mapping information on important sorghum traits and we need strategic targeting of appropriate improvements. The decisions as to which improvements are appropriate will vary according to the targeted user, and will take into account the resources available, the value of the improvement, relative ease of the improvement, and biosafety factors.

This article describes a large variety of potential improvements that could be made in sorghum with assistance from the full gamut of biotechnologies. In addition, it discusses current limitations in improving sorghum via biotechnology. These limitations can be overcome by appropriate research and development, and they must be overcome if sorghum is to continue making significant contributions to world agriculture.

MOLECULAR BIOLOGICAL TOOLS AND TARGETS FOR SORGHUM IMPROVEMENT

Any crop improvement programme can utilize new DNA-based technologies at two very basic levels: enhanced breeding technology and genetic engineering. The use of DNA markers to augment traditional plant breeding approaches is currently ongoing in many crop species and can greatly increase both the rate and scope of such improvements (Tanksley et al., 1989; Paterson et al., 1991). Genetic engineering is also underway with a few crop species, although some technical limitations remain. Taken together. these two new tools will allow improvements in sorghum beyond levels possible with any other approaches. Yields will be increased, production costs will be lessened, and new products will be generated. In the increasingly competitive environment of international agriculture, those crops that do not keep pace with this tremendous rate of improvement will be marginalized or abandoned.

Enhanced sorghum breeding: parental choice, line identification, and marker-assisted selection. A wide array of DNA marker technologies, including restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), can be used to characterize genomes. The uniquely powerful attributes of DNA marker technology applied to a higher plant like sorghum include the vast numbers of markers that can be studied, the lack of epistatic interaction between markers, and the ease of the assays for polymorphism. DNA probes were used to generate the first detailed genetic maps of sorghum (Hulbert et al., 1990; Binelli et al., 1992; Whitkus et al. 1992; Melake-Berhan et al., 1993; Chittenden et al., 1994; Pereira et al., 1994; Ragab et al., 1994; Weerasuriya, 1995 ), and these combined maps now contain several hundred linked markers.

DNA markers can be utilized to characterize sorghum lines for their suitability as parents in a breeding programme. For instance, lines containing DNA polymorphisms associated with particular desirable characteristics (e.g., a specific

heterotic group or fertility factors. yield components, etc.) can be identified in a collection once enough correlations and cosegregations of these traits and markers have been established (Messmer et al., 1991;Tao et al., 1993; Vierling et al., 1994). Unexpected sources of diversity (or homology) can be identified. For example. we have found that the aethiopicum, arundinaceum and virgatum races of S. bicolor ssp. verticilliflorum, despite their weedy phenotypes, primarily contain allelic variants found in cultivated S. bicolor ssp. bicolor from the same regions. In contrast, the agronomically-adapted Chinese sorghums are very polymorphic compared to other cultivated sorghums, and should, therefore, provide an excellent source of diversity for improvement of African and Indian sorghums (Oliveira and Bennetzen, unpublished).

The vast number of potential DNA markers. and high levels of DNA polymorphism, essentially guarantee that any two sorghum lines can be differentiated. This allows unambiguous identification (and protection) of any distinct sorghum cultivar. Moreover, the origins of the various chromosomal regions of any sorghum line can be traced back to the appropriate parents (Gebhardt et al., 1989; Messmer et al., 1991). This approach provides insights into the molecular basis of successful breeding programmes, and associates particular agronomic traits with particular chromosomal regions.

Marker-assisted breeding will be an especially powerful use for DNA markers. Once a trait is mapped within a particular line, then that trait can be efficiently introduced into any other line by following flanking DNA markers rather than the phenotype itself. This will be particularly useful for traits which are difficult to assay due, for instance, to poor penetrance or inconsistent exposure in field tests. Moreover, any number of traits can be followed at once. including quantitative trait loci and traits that would show epistasis if assayed phenotypically. These will be particularly useful for stress resistance traits where the use of DNA markers will allow pyramiding of multiple different resistance genes in the same line.

Weerasuriya (1995) has recently mapped various traits in a recombinant inbred (RI) population of sorghum, including time to maturity, plant height, and resistances to Striga, rust, and anthracnose. Nine different DNA segments account for anywhere from 25 to 100% of the variation for each of these five traits in these RI lines, and markers for these nine chromosomal segments could be used to introgress all of these traits into a new background at the same time. Moreover. DNAs not linked to the desired traits could be rapidly removed from such a backcross breeding programme by selecting those progeny that have the least undesired DNA from the non- recurrent parent. This can shorten a backcross introgression programme by four or more generations, eliminate the need for field assessment of every trait in almost every generation, and allow selection of rare recombinants that will have the least linkage drag on undesired traits. It is difficult to over-emphasize how routine this technology is once the traits are mapped; a single investigator could easily introduce ten to forty improved alleles into a series of different backgrounds in just a few years.

Genetic engineering of sorghum. In these early days of plant genetic engineering, our chief limitation remains identification of sources (and clones) of genes that could provide improved performance to a crop species. Proposed applications of genetic engineering to crops fall into the general categories of improving plant stress resistance, improving crop quality, decreasing costs of production, and expanding the products that may be obtained from a crop. In no case have durable gains from this approach been demonstrated in any crop, but the potential is enormous.

Technologies now exist for the cloning of genes from any organism, for transformation of these genes into sorghum (Casas et al., 1993) and other crop plants, and for modification of these genes so that they are expressed at levels and in tissues targeted by the genetic engineer. The maintenance of desired levels of transgene expression in multiple progeny generations has been ambiguously established, but techniques are under development that either target the transgene to a "natural" genomic expression site (Paszlowski et al., 1988) or add "insulating" elements (Goldman, 1988) that should protect the transgene from position effects and/or sense suppression (Finnegan and McElroy, 1994) phenomena. This technical limitation will be overcome, and the time is now right to begin to test the potential of genetic engineering for sorghum improvement.

Several genes are available that have been shown, or postulated, to provide resistance of a broad range of plants to various biotic and abiotic stresses. Crystal protein genes from strains of Bacillus thuringiensis provide defense against some insects (Gasser and Fraley, 1992), as do other genes that encode proteins which interfere with insect digestion (e.g., proteinase and alpha amylase inhibitors). Similarly, engineered constitutive- and/or over-expression of various pathogen-induced plant gene products (e.g., chitinases, glucanases) have been shown to inhibit the pathogenicity of various fungi and bacteria (Zhu et al., 1994). Viral pathogenicity has been attenuated, or even eliminated, by coat protein expression and/or sense suppression in transgenic plants (Smith et al. 1994). Disease resistance genes have now been cloned from several plant species (Johal and Briggs, 1992; Martin et al., 1993; Bent et al., 1994; Jones et al., 1994; Mindrinos et al., 1994), and these may be engineered to possibly provide more effective resistance.

Genetically engineered abiotic resistances have not yet been definitively demonstrated in field tests, but genes from species highly adapted to adverse environmental conditions will be transferred into a broad range of crops and tested for effect (Duman and Olsen, 1993). The use of DNA marker technology to map quantitative or qualitative traits that provide resistances to abiotic stresses, as with biotic stresses, will allow these genes to be cloned and transferred from stress-tolerant species to stress-sensitive crops. Engineering for controlled expression of genes that are induced by drought, heat or cold offers significant promise. As with engineered biotic resistances, the field efficacy of these traits will need to be proven; it is likely that some of these changes will have undesirable negative effects on yield in the absence. of heavy stress exposure.

Crop quality can be modified in a number of ways, often by alteration of single steps in intermediary metabolism. In sorghum, engineering of higher levels of expression of amylase genes in the endosperm could be relatively easily accomplished, and may improve sorghum's malting properties. Genes could be added that would improve the nutritional balance of sorghum seed proteins, or alter the level and/or types of starches and oils. Forage properties could be altered by modification of the composition of lignin and other components of the leaf and stalk. Versions of many of these alterations can be, and have been, accomplished by classical mutational breeding. However, genetic engineering can target these changes more precisely, and can be used to alter physiological pathways that may be recalcitrant to standard genetic manipulation due to the presence of duplicated or pleiotropic loci.

Decreased costs of crop production could be accomplished by a number of engineering approaches, including attainment of higher levels of stress resistances that would decrease the need for pesticides, irrigation, etc. In some agricultural systems, a broader array of engineered herbicide resistances (Gasser and Fraley, 1992) will allow the farmer to choose the herbicide that costs the least or has the least negative effect in his or her environment. New methods of generating male-sterile lines could lead to a decrease in the cost of hybrid seed. In the very long term, sorghum and other grass crops may be engineered to productively associate with nitrogen-fixing microbes, thereby decreasing the requirement for addition of exogenous N fertilizers.

Perhaps the most exciting, and eventually most important, future outcome of plant genetic engineering will be in the modification of crops to yield wholly new products. Many of the industrial processes that support our societies have traditionally been accomplished using manual labour, simple machines and chemical processes. Natural selection has created biological systems that out-perform all of these non-living systems, and enzymology is replacing many older techniques for production. Plants are incredibly efficient machines, use an inexpensive energy source (sunlight), and require relatively little maintenance. In the near future, plants will be used to produce pharmaceuticals, chemicals, polymers and biofuels. Although limitations exist in the production of cost-effective levels of these compounds in plants, and in efficient purification from plant tissues, these same limitations initially impinged on the use of microbial fermentation to

Biotechnology for sorghum improvement produce such materials. As the efficiency of the production of such new-products in planta increases, the much lower cost of field growth of plants compared to growth of microbes in a fermentation facility should win the day. The potential of plants as factories is almost boundless, and these non-traditional uses of agriculture may become the greatest sources of farm income within a few decades.

LIMITATIONS IN THE APPLICATION OF BIOTECHNOLOGY TO SORGHUM

The central limitations to improvement of sorghum using biotechnology are the dearth of available information and the lack of resources dedicated to sorghum study and improvement. Despite the relatively small amounts of attention that

covering the whole range from sorghum to maize to rice to wheat, have very similar gene content and regions of conserved gene order (Hulbert et al., 1990; Ahn et al., 1993; Devos et al., 1993; Melake-Berhan et al. 1993; D'Hont et al., 1994; Kurata et al., 1994; Pereira et al., 1994). This means that genes can be efficiently (and comparatively) mapped in any grass species using an anchor set of grass DNA markers and that any grass species can be used as a source of genes for sorghum improvement (Bennetzen and Freeling, 1993;Bennetzen, 1995). Hence, one may be able to transfer the cold tolerance of barley or the seedling vigour of maize to sorghum once the appropriate genes are mapped and cloned in these species.

Because rice, barley, maize and sorghum (for instance) contain nearly the same gene content by homology criteria, the different agronomic properties of these species must be due to allelic molecular biologists have paid to .sorghum, the last five years have seen tremendous advances in mapping (Hulbert et al., 1990; Binelli et al., 1992; Melake-Berhan et al., 1993; Chittenden et al., 1994; Pereira et al., 1994; Ragab et al., 1994), diversity analyses (Tao et al., 1993; Vierling et al., 1994), and transformation (Casas et al., 1993). With this full set of molecular tools now in hand, biotechnology can be applied to sorghum once appropriate improvements are identified.

A large number of useful DNA markers have been identified and mapped. and there is little need for more to be acquired from sorghum. However, there are too few fingerprinted lines, mapped traits and mapped populations in sorghum. The populations that have been mapped were often too small to provide precise mapping information. Important single gene and quantitative traits need to be placed on the sorghum genetic map (Oh et al., 1994; Weerasuriya. 1995 ), and the traits need to be screened under a variety of environmental circumstances, and from a number of parental sources. Such comprehensive studies will be needed to identify regions of the sorghum genome that enhance productivity and to identify the lines that have the superior alleles within these regions.

This important gene identification and mapping process can be greatly assisted by use of information and markers from other grass species. Recent studies have shown that the grasses, variation in these genes. Analyses of wild relatives of maize (Doebley and Stec, 1991) and wild relatives of sorghum (Oliveira and Bennetzen, unpub.) indicate that very few genetic changes are needed to bring about these major differences in morphology, physiology, and growth habit. Thus, the whole range of grass species can be used to identify superior alleles of specific genes, and these "superior" genes will often function effectively when transferred into other grass species (Bennetzen and Freeling, 1993;Bennetzen, 1995). For example, quantitative loci influencing seed weight (Fatokun et al., 1992) or plant height (Pereira and Lee, 1995) have been mapped to orthologous positions in related plant genomes. Their similar phenotypic effects and map positions suggest that these genes may be "allelic" and the genetic engineer can choose whichever allele preferred for the crop modification process.

In contrast, Weerasuriya (1995) identified a rust- resistance gene in sorghum that maps to a position where no rust-resistance gene has ever been mapped in the collinear maize genome. This suggests that the mapped sorghum gene is one that either does not exist in maize or has not been genetically uncovered in maize. Hence, engineering of this gene into maize, or of the maize rust-resistance genes into sorghum, may provide a novel source of resistance not previously available to either species.

The mapping of agronomically significant genes from multiple grass species will provide the basic information that can be used to plan enhanced breeding strategies using DNA markers. The cloning of these same genes should provide the raw material for the improvement of sorghum by genetic engineering. Although this work needs to be actively pursued in sorghum, one also should take advantage of genes and mapping information from other grass species which can be directly used to improve sorghum.

Given the limited resources that have been brought to sorghum research, the gains in sorghum productivity by traditional breeding and the recent successes in molecular technology development have been impressive. Biotechnology offers a wide array of possible improvements for sorghum. However. these modifications vary tremendously in how difficult (and expensive) they would be, what level of improvement they might produce, who they would assist, how fast they can be attained, and what possible negative impacts they might provide. Approaches to improvements in abiotic stress resistance, or nitrogen fixation/ utilization, are only in their first stages of analyses and will probably be long in coming despite their major importance. New herbicide resistances could be easily engineered into sorghum, but these would not be of use to a subsistence farmer who cannot afford herbicides and could also yield an exceptionally negative outcome if cross-pollinated into weedy sorghum relatives. Expression of genes that encode anti-microbial compounds could be simply accomplished in sorghum, but their effectiveness is not clear (and may be highly variable). Hence, sorghum researchers would be wise to marshal their limited sources for less difficult or tenuous targets.

One area in which biotechnology could immediately enhance sorghum productivity would be in the areas of parent identification and marker-assisted breeding. A single gene that provides good field resistance to Striga has been identified and mapped (Weerasuriya, 1995) and could be efficiently introduced into a large number of elite sorghum lines with a minimum of effort. Other important traits are being mapped, and their sources identified, which should allow this technology to be applied in the near term. Specific "off-the-shelf" genetic engineering, like B. thuringiensis

crystal protein synthesis. could improve sorghum, although the durability of such changes is not clear. In all cases, the researchers need to have first undertaken a thorough cost/benefit/risk/time frame analysis in which the potential user is clearly identified. One would expect that projects designed to assist the subsistence farmer will often be very different from those formulated to enhance production agriculture, although some very key improvements could benefit a broad range of users.

BIOSAFETY CONSIDERATIONS

Any process that improves a crop will lead to alteration in the ways and levels at which that crop is produced and utilized. Changes brought by biotechnology will be no different in this regard to those resulting from traditional plant breeding. Moreover, even the horizontal gene flow associated with some genetic engineering applications has natural precedents.

There are, however, properties of biotechnology-based improvements that will be clearly distinguishable from improvement via traditional crop breeding. First, biotechnology will greatly increase the rate of crop improvement. New varieties, with greater-than-standard increases in value, will come on line more frequently. Second. genes will be brought into crops from various evolutionary sources, and in altered forms. more often than would occur in standard plant breeding. And third, the changes made via biotechnology will be relatively precise and well-characterized.

Wide dissemination of outstanding new materials with a narrow genetic base could lead to a decrease in the variability present in the agricultural milieu, as has been seen when improved varieties replace traditional landraces. Field populations with a narrow genetic base can be susceptible to catastrophic production losses in the presence of new biotic or abiotic stresses. Because biotechnology will yield exceptionally superior cultivars very rapidly, this technology might lead to a more rapid loss of field diversity. However, a decreased genetic base for improved varieties is not an obligatory outcome of crop improvement; rather, it is caused by poor planning and lack of vision by those organizations that produced the improved varieties. Any thoughtful agricultural programme would target field diversity as an essential component of dependable farm productivity.

Biotechnology can greatly assist the process of acquiring and efficiently using diversity in sorghum or any other crop. The use of DNA markers will efficiently find allelic diversity, and marker-assisted selection can allow particular superior traits to be efficiently introduced into many backgrounds concurrently. Hence, the plant breeder will now have the resources to release many more micro-adapted, diverse-background cultivars than could have been possible when breeding relied more heavily on field tests and slow crossing/selection programmes.

The entry of wholly new genes into crop plants, via genetic engineering, holds forth much of the promise of biotechnology and has raised the most fears of possible problems. Transfer of genes between distantly related-species does occur in nature (Syvanen, 1994) but this horizontal gene transmission is fairly infrequent. However, plant and animal breeders have traditionally performed crosses between closely-related species, providing the basis for much agricultural improvement. This intermixture of thousands of genes from two different species has occasionally given rise to a problematic outcome (for instance, the Africanized or "killer" honey bee now spreading across the Western Hemisphere), but in most cases the results have been beneficial. The "genetic engineer" is usually adding only one or a few genes to a species, and usually knows the function of the engineered gene or genes. Hence, the unforeseen dangers of plant genetic engineering should be less than those of traditional approaches using varieties bred from wide crosses.

Evaluation of the possible negative effects of a given crop improvement needs to be made on a case-by-case basis. Traits engineered into sorghum are likely to be transferred to its weedy relatives, so one should avoid adding genes to sorghum (e.g., herbicide resistance) that one does not wish to see acquired by these weeds. Other traits, like improved Striga resistance, seed protein quality, or superior malting potential, are not likely to have any negative effects if transferred to sorghum relatives. Hence, it is a tremendous positive factor that possible problems generated by biotechnological advancement of sorghum can be foreseen and avoided at the outset. This is not nearly so true in traditional crop breeding programmes.

It is almost certain that biotechnology will yield some problems, as have all other new technologies. For instance, biotic stress resistances engineered into sorghum may create an environment in which pests or pathogens evolve the ability to overcome these resistance mechanisms. To my mind, this will be particularly worrisome with coat protein mediated vital resistance or B. thuringiensis toxin resistances based on single crystal protein genes. This does not mean that such approaches should not be attempted, however. Instead, the efficacy of particular targeted improvements needs to be constantly monitored, and agencies involved in crop improvement need to resist the lazy/greedy temptation to put all of their eggs in one basket.

The same Luddite arguments used against biotechnology in agriculture could have been used to block the utilization of antibiotics in human health care or to prohibit the wide-crosses and other crop breeding technologies that yielded the green revolution. Yet without these technical advances, the quality and quantity of human life on this planet could not be maintained at any where near its current levels. The directed nature of biotechnological changes allows biorational judgment in deciding which changes have the best benefit:risk ratio. When problems do arise, they will be dealt with using the full set of agricultural tools, including biotechnology.

CONCLUSIONS: BIOTECHNOLOGY AND THE FUTURE OF SORGHUM

Sorghum is one of the world's great crops and plays an essential role in those regions of the world where heat, drought and poor soils make other crop production systems untenable. Biotechnology can advance sorghum improvement at a tremendous rate, both by providing more powerful and efficient tools to the plant breeder and by providing access to new genetic functions. Other crops will also be greatly improved using biotechnology, including the acquisition of some sorghum traits by direct gene transfer. Hence, sorghum must participate in the biotechnology revolution or face abandonment as a significant agricultural contributor.

The utilization of DNA markers to assist sorghum breeding could be initiated immediately; the tools and preliminary information are available. Target priorities need to be set, and introgression of genes for biotic stress resistances into already adapted cultivars would be a good choice. Genetic engineering also holds great potential, and simple additions of genes that could provide malting or biotic stress resistance characteristics could also be undertaken. The future of sorghum, and other crops, as factories for the production of a wide range of new products offers enormous long-term promise.

Many of the limitations to biotechnological improvement of sorghum (long term transgene stability, possible negative outcomes of horizontal gene transfer, identification and isolation of superior genes for improvement, etc.) also apply to all other crops. Hence, sorghum will benefit from the extensive studies that will be performed on other crop plants. Careful consideration of the needs of sorghum producers and users should allow identification of safe, cost-effective and highly valuable applications of biotechnology to sorghum improvement.

ACKNOWLEDGMENTS

The preparation of this manuscript was supported by the McKnight Foundation and USDAINRIGP award 94-37300-0299.

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