African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 6, Num. 3, 1998, pp. 303-315

A. M. Thro, W. Roca1, C. Iglesias¹, G. Henry² and S.Y.C. Ng³

Cassava Biotechnology Network, c/o Centro Internacional de Agricultural Tropical (CIAT), Cali, Colombia
¹Biotechnology Research Unit, Cassava Breeding, and Genetic Resources Unit, CIAT
²CIRAD, Montpellier, France
³International Institute of Tropical Agricultura (IITA), Ibadan, Nigeria

(Received 17 November, 1997; accepted 7 May, 1998)

Code Number:CS98032
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ABSTRACT

Cassava (Manihot esculenta Crantz) is a tough and productive survival crop of smallholder farmers in the tropics. It is often grown where other crops fail. Research can make important contribution to food security and economic development in cassava-growing areas. This can be enhanced through in-vitro propagation methods for this crop, due to its slow vegetative propagation. For useful biotechnology innovations to reach smallholder cassava farmers, a combined effort is essential, with participation of farmers and other partners in research and development. The Cassava Biotechnology Network combines forces to mobilise biotechnology as a tool for enhancing cassava's value for food security and as a means of access to economic development. In-vitro biology methods are essential to safe and efficient conservation and exchange of cassava diversity. Transgenic methods, now nearing the stage of first field trials, may offer a means of adding disease resistance or enhanced crop quality characteristics to existing preferred varieties.

Key Words: Cryopreservation, genetic diversity conservation, genetic transformation, Manihot esculenta Crantz, micropropagation

Le manioc (Manihot esculenta Crantz) est une culture résistante et productive de survie de petits fermiers dans les tropiques. Il pousse souvent là où les autres cultures échouent. La recherche peut faire une contribution importante pour la sécurité alimentaire et le développement économique dans les zones de la culture du manioc. Ceci peut ê accru à partir des méthodes de propagation en vitro pour cette culture suite à sa propagation végétative lente. Pour des innovations biotechnologiques utiles afin d'atteindre les petits fermiers producteurs du manioc, un effort combiné est essentiel avec la participation des fermiers et des autres partenaires en recherche et developpement. Le Réseau Biotechnologique du Manioc combine les forces pour mobiliser la biotechnologie comme un outil pour accroître la valeur du manioc pour la sécurité alimentaire et comme un moyen d'accès au développement économique. Les méthodes biologiques en vitro sont essentielles pour une conservation sûre et efficiente et pour l'échange de la diversité du manioc. Les méthodes transgéniques s'approchant maintenant au stade des premiers champs d'essais peuvent offrir un moyen d'augmenter la résistance à la maladie ou d'améliorer les caractéristiques augmentées qualitatives des variétés actuelles préférées.

Mots Clés: Cryoconservation, conservation de la diversité génétique, transformation génétique, Manihot esculenta Crantz, micropropagation

Cassava (Manihot esculenta Crantz) is the most important locally-produced food in a third of the world's low-income, food-deficit countries, especially in Africa (FAO/GIEWS, 1995). Cassava's harvest of starchy roots and high-protein leaves is reliable even in extreme conditions where cereals fail. It is especially important in times of social insecurity and war, when other crops are stolen or destroyed. For women heads of household, struggling to feed their families without social safety nets, cassava's dependability is critical.

Cassava also provides economic opportunity in poor rural areas. Using simple technology, the roots are processed to flour and starch. Markets for these are growing as cities demand breads and other convenience foods. Yet cassava farmers remain poor.

Rural poverty has many causes. Some are social and economic (see Brett, 1992) ; others are related more directly to agricultural productivity: poor soils, irregular rainfall, varieties with poor stability, low inherent yield potential or poor market quality. Research can help tackle these problems. For example, cassava yield and yield stability can be improved for marginal regions through genetic improvement of drought tolerance, photosynthetic efficiency, and resistance to pests and diseases. Improved keeping quality (cassava must be processed within 24 hours of harvest) could open new market opportunities for cassava and greatly reduce crop losses.

In the case of cassava, research has unique linkages to development objectives for the some of the poorest rural areas of the tropics (Thro et al., 1998a, in press). In addition to being adapted to marginal environments where other crops are risky, cassava has a high potential energy production per land area. It offers useful management flexibility to the farm family, via relatively wide windows for planting and harvest, suitability for intercropping or monocropping in different environments from humid to sub-arid, in-ground pre-harvest storability, built-in protection from theft and animal damage (Chiwona-Kaltun et al., 1997; Kapinga et al., 1997), and suitability to be consumed at home or sold, in fresh or processed form. Some of cassava's disadvantages stem from the fact that it is unknown outside the tropics. It therefore has no Northern heritage of strategic research on which to draw. As a result, in more favoured areas, cassava production may be unattractive, and cassava remains relatively more important in poor unfavoured areas where there are few other crop alternatives. Such areas are occupied predomi-nantly by small scale farmers.

World market reforms will affect small-holder cassava farmers. Subsidies to wheat and maize imports will decrease, and cassava will compete more favourably in the new markets. Appropriate research will enable cassava farmers to supply a low priced, high quality product to take advantage of resulting new market opportunities. An upgraded cassava crop with vibrant markets will enhance productivity and rural income in cassava-dependent areas. This will contribute to food security, poverty alleviation, equity and environmental protection (a valuable crop is an incentive for good land husbandry). In-vitro technologies will be a part of the research and development effort to enhance cassava's value for food security and rural development .

The Cassava Biotechnology Network (CBN) was founded in 1988 by the Centro Internacional de Agricultural Tropical (CIAT) in South America and the International Institute of Tropical Agriculture (IITA) in Africa, with a handful of laboratories in Europe and North America. The network's initial vision, still valid today, was to enlist leading laboratories in these regions for cassava biotechnology research around a common strategic agenda, help use existing research investment cost-effectively, and stimulate research in cassava-growing countries. Initial thrust was concentrated on developing biotechnology tools for cassava research. Under a new partnership with the Government of the Netherlands in 1992, CBN accepted the challenge of integrating the farmer into the research planning process. This led to development of a broader vision within CBN. To help assure desired impact of biotechnology research, CBN's strategy is to foster the integration of biotechnology tools (or awareness of their potential) in appropriate ways throughout the cassava research and development cycle, from needs assessment through strategic and applied research, to technology transfer, impact assessment, and feed-back to research (Table 1). CBN also works to create linkages between biotechnology research, applied cassava research, and farmers (Table 2).

TABLE 1. Partial list of CBN collaborators in needs assessment, strategic and applied research on identified needs, and technology transfer

TABLE 2. Cassava Biotechnology Network collaborators in cassava strategic research

In needs assessment, for example, CBN deals directly with cassava farmers in rapid case studies conducted with farm families in fields, villages and homesteads (Thro et al., 1994; Henry and Howeler, 1995; Thro et al., 1997). Through its members, CBN is also linked to long-term projects involving farmers and researchers working together (Iglesias and Hernandez, 1997) (Table 1). For example, in semi-arid Northeast Brazil, CIAT, IITA, the national cassava programme CNPMF, and "COPALS" (farmer research committees) are developing integrated pest and crop management systems.

Such projects provide excellent information on farmers' perspectives. Information from farmers and rural processors is reviewed with national and international disciplinary experts to identify farmer priorities where biotechnology may offer significant assistance. This step also looks for new insights and creativity that appear when broad understanding of farmers' underlying priorities is combined with a grasp of the innovations that biotechnology tools can make possible. Priorities from this process are published for further comment and debate until the next cycle.

Current cassava research priorities, after several cycles of discussion with farmers and researchers, include rapid propagation of planting material, environmental adaptation (including plant protection), economic and nutritional value, and keeping quality.

Two basic "ingredients" are required to develop improved varieties of cassava, or any other crop: genetic diversity (the "raw material"), and adequate field testing. Put very simply, anything that increases access to genetic diversity or efficiency of field testing will allow cassava breeders to make faster and further progress. In-vitro biology for cassava is most important for the first "ingredient": it gives cassava breeders and farmers better access to genetic diversity, and is a part of methods to create new forms of genetic diversity for cassava.

Through use of tissue culture, disease-free plantlets conserve cassava genetic diversity without the long-term losses of field collections, and genetic diversity is safely shared internationally (Roca and Nolt, 1989). Tissue culture can potentially allow rapid multiplication of healthy planting material of desired varieties (Roca et al., 1991) (always the farmer's first concern). Genetic engineering (or genetic transformation) increases diversity by altering expression levels of existing traits or adding new traits (new genes) to otherwise superior varieties.

Special importance of in-vitro biology for cassava. For most crops, planting is done with seeds, which are also used for conservation and exchange of genetic diversity. For cassava, however, propagation is vegetative, using lignified stem sections, or "stakes". This is because cassava is an outbreeding species that experiences severe loss of vigour if inbred to obtain true-breeding seed. Many clones produce vigorous seed useful for selecting new genotypes and for preserving genes per se, but not for preserving or multiplying desired varieties (Henry and Iglesias, 1993).

Cassava's vegetative propagation is a mixed blessing. The lignified stakes used for planting can survive in spite of delayed rains, when seedlings would die. But other consequences are key limitations to the improvement of cassava-based agriculture (Henry and Iglesias, 1993):

Just how serious this bottleneck is, is seen in a comparison with cereals such as maize or rice. Cassava's growth cycle is commonly 12 to 18 or even 24 months. One harvested plant produces enough stakes for about 10 more. One plot of cassava produces enough planting material for about 10 similar-sized plots. Cereals, in contrast, commonly have growth cycles of 3 to 6 months and produce enough seeds to multiply the original area by 100 times or more. To plant one hectare, a farmer needs 100 kg of cassava stakes, compared to 25 kg of maize seed (Henry and Iglesias, 1993). The bulk and weight of stakes is a major logistical consideration. Adding to the farmer's challenges is that in most environments, cassava stakes cannot be stored but must be replanted soon after harvesting.

Clearly, any technology will have a major impact if it can provide disease free planting material and alleviate cassava's multiplication constraints at the farm level. Micropropagation is being explored locally for these purposes in the near term. Transgenic techniques are expected to begin making a contribution in about five years (Thro et al., 1998b, in press).

Germplasm conservation and exchange. The world cassava collection maintained at CIAT contains over 5000 genotypes, in the field and in an active in-vitro genebank. IITA maintains 1,800 clones in the field and 700 in-vitro, mainly African landraces. Each centre ships from 3000 to 4000 virus-tested vitro-plantlets per year to collaborators (e.g. Ng and Ng, 1997). This has contributed to the selection of varieties for multiplication and release in several countries in Africa (IITA, 1997a), 12 varieties in Latin America, and five in Asia (CIAT, unpub. data).

Slow-growth conditions reduce subculture requirements to once each 12 to 24 months, depending on the genotype (Roca et al., 1991; IPGRI/CIAT, 1994). Genetic stability of the clones is not affected by up to ten years in in-vitro culture under these conditions (Angel et al., 1996). During a three-year pilot project involving the first 100 accessions to be entered into the in-vitro genebank at CIAT, no clones were lost completely and only about 35 clones were lost from 1 to 3 of 5 total replicates. During the same period, the field collection at CIAT lost at least one of the 100 clones completely and from 1 to 4 replicates of 61 other clones (IPGRI/CIAT, 1994). These data support the strategic value of an in-vitro collection, particularly if the collection is not duplicated at another location. Epperson et al. (1996) found that cost of maintaining the field and in-vitro collections were comparable given Colombian costs for skilled labour; higher labour costs would favour the field collection.

The in-vitro genebank technology for cassava has been successfully adapted by over 20 countries, for conservation of national cassava diversity and exchange with other countries. The ability of national programmes to sustain funding for in-vitro cassava genebanks is currently more problematic than the technology. Low cost of skilled labour is often an advantage in these countries, but cost and erratic supply of media components and electrical power are obstacles.

Cryopreservation of cassava would permit long term storage of cassava genetic diversity, with lower recurrent costs and without the need for special growth room facilities. In work at CIAT, initiated in collaboration with IPGRI, it is now possible to hold cassava shoot tips in liquid nitrogen for as long as one month. Plant recovery after freezing ranges from 20% to 70 % depending on genotype. Factors contributing to success have been high illumination of donor cultures, preculture of shoot tips, a low concentration semi-solid cryoprotection media, and rapid freezing (Escobar et al., 1995). Before the technology can be used and transferred, a pilot study is needed to assess both costs and the long-term viability and genetic stability of the cryocultures.

Associated with high costs of reagents and facilities, are post-flask losses of vitro plantlets. Particularly in the poorer countries, and in remote areas where cassava is most needed, shipments of new varieties for testing often experience high or total losses during the hardening stages when vitro-plantlets are transferred from test tube to stake propagation nursery. Low cost methods have been used successfully in China, Cameroon, and Congo (Guo and Liu, 1995; Zok, 1993; Mabanza et al., 1995). A simple, low input post flask management system for acclimatisation of cassava in vitro plants (Ng et al., 1994) is used successfully in many countries in Africa, and is constantly modified based on feedback from national programmes. A collaborative study involving several university and national programme partners is now underway in Zimbabwe and Mozambique to examine factors affecting post-flask survival. The objective is to develop robust, low-cost methods that can be used successfully when infrastructure, labour, and even water are limiting.

Clean planting material for conservation and production. Thermotherapy techniques (Roca et al., 1991) are used to provide clean planting material of cassava. These are essential for germplasm collections and for material to be exchanged with other countries. In cassava production, positive yield responses after thermotherapy to obtain clean planting material have been shown in Cuba (Garcia et al., 1993), Cameroon (Zok, 1993), and Peru (Delgado and Rojas, 1993). Negative responses in Brazil (M. Lourenco, Lorenz Company, pers. comm. 1994) may have been due to associated loss of beneficial microorganisms. This can be rectified through inoculation. A more serious problem is rate of reinfection. Although in-vitro clean up of susceptible varieties may be a valuable short-term emergency measure, it is essential to understand all costs and the disease situation in the field before recommending it as a long-term strategy for providing clean planting material. Reinfection has been almost absent in specific low-disease pressure situations, making this an effective method; but under high disease pressure, rates are high (Akano et al., 1997) and can be so high that genetic resistance is the only economical long-term solution (Thresh et al., 1997).

Rapid varietal propagation: In-vitro meristem culture. In-vitro culture of meristems can permit multiplication of desired cassava varieties thousands of times faster than present methods (Roca, 1984). A partnership of IITA and World Vision successfully used micropropagation to produce large quantities of propagation material of selected clones for shipping to war-affected areas in Angola and other African countries (IITA, 1997b). Similar effort is in progress to re-introduce germplasm to countries such as Liberia, Sierra Leone and Ecuador in collaboration with the national programmes and USAID.

In vitro culture is also being tested in pilot projects aimed at developing robust low-cost systems, as well as local skills in management of logistics and costs. Successful post-flask management (low cost and adequate survival rates) is a key to the success of these projects. During the post-flask period, plantlets must adapt to differences in humidity, light, and growth media, and especially aseptic conditions.

One pilot project, in collaboration with NARO in Uganda, is testing feasibility of tissue culture for multiplying resistant cassava varieties to combat a severe viral epidemic that has wiped out many traditional cassava varieties in the country. The tissue culture operation will be integrated into the existing variety multiplication scheme of the national cassava programme, with district extension agents and farmer groups. Another set of pilot projects, in Colombia (in collaboration with the national programme CORPOICA, a farmers organisation ANPPY, and the NGO FIDAR), will explore feasibility of local production of cassava tissue culture plantlets as a household micro-enterprise or project for farmer co-operative nurseries. One project is in an area where bacterial blight is endemic. The characteristics of the preferred local variety, which is susceptible to bacterial blight, have been difficult to recover in a resistance breeding programme. Use of good cultural practices to maintain healthy planting material is not practical because small-scale farmers in this area commonly are renters, renting land for less than 12 months (Thro et al., 1997). Since market demand in the area is high, tissue culture can help improve their production and income while other social and biological solutions are sought.

Rapid varietal propagation: Bioreactors. Somatic embryogenesis could form the basis for a system of encapsulated "somatic seed" of desired cassava varieties (Henry and Iglesias, 1993). Recent development of embryogenic suspension cultures of cassava (Taylor et al., 1996) makes this approach technically feasible. Key questions include (i) level of sophistication in management of planting material; (ii) effects on production costs, which must remain low for cassava to remain competitive in contemporary agriculture; and (iii) germination and establishment of synthetic seeds in adverse conditions where cassava is grown.

The power of the new in-vitro technologies for cassava multiplication, makes it ever more critical that biotechnology research in cassava integrates the participation of farmers, agronomists, and socio-economists. An integrated approach is essential. Varieties must be proven reliable in farm and market before funds, time, and credibility are invested in their mass propagation. Generally, several preferred clones should be multiplied, to permit farmer selection and genetic diversity. Knowledge of farming systems, processing, and market systems will be essential for successful use of mass propagation, and especially for assessing the social and economic costs and benefits of novel propagation systems (bioreactors/ synthetic seeds).

Genetic transformation. Four groups have confirmed regeneration of genetically transformed cassava plants (Sarria et al., 1995; Li et al., 1996; Raemakers et al., 1996; Schöpke et al., 1996), and several other groups (e.g. Ohio State University, USA; Centro de Ingenieria Genetica y Biotecnologia, Cuba) are working independently to develop similar or different methods. The confirmed transgenics have been produced using both Agrobacterium and microprojectile transformation systems, with both secondary somatic embryos and embryogenic suspension cultures as the target regeneration systems. The clones used as models in most cassava genetic transformation work to date are TME 60444, provided by IITA, and M Col 22, a Colombian landrace from the world collection held at CIAT, but recent experience suggests that methods can be developed for a wide range of cultivars (Thro et al., 1998a, b in press). A recent review by Raemakers et al. (1997) provides a thorough description of the status of this research. The diversity of successful approaches implies that improvements in efficiency and robustness can be obtained. Major effort will now be directed at the introduction of genes of agronomic interest. Extension of genetic transformation systems to a range of important varieties, and identification of tissue-specific promoters, are high priorities. Transgenic cassava carrying genes of interest should be available for field trials within one to three years, in countries with biosafety policies in place. In Africa, countries currently most advanced in the process of developing national biosafety regulations, i.e., closest to readiness for testing transgenic cassava, include Nigeria, Zimbabwe, Kenya, and South Africa.

Transgenic approaches to disease and pest resistance for cassava. Genetic transformation is expected to contribute first to providing new sources of disease and pest resistance. A new form of African mosaic virus (ACMV) has wiped out all traditional cassava varieties in Uganda (Otim Nape et al., 1997). New varieties with a high level of tolerance are holding up during the ACMV epidemic and are of "acceptable" quality, but are not yet the preferred types. Yet they have taken breeders many years to develop. Common cassava mosaic virus (CCMV) causes production losses in north-eastern Brazil, losses that are currently being addressed experimentally in a pilot project by a joint public-private venture for in-vitro clean-up.

In the long run and for the smallest farmers, genetic resistance of a type that could be readily added to existing varieties, via transgenic techniques, offers advantages. Such a technology would cost the farmer least to adopt, with minimal change to any other parameter of the farming system. Genes now available for cassava include a viral coat-protein (CP) gene imparting resistance to common cassava mosaic virus (CCMV) and a disfunctional viral replicase gene imparting resistance to African cassava mosaic virus (ACMV) (SchÜpke et al., 1993; Thro et al., 1998a in press). Both genes have been incorporated into the African cassava variety TME 60444 through genetic transformation; CCMV CP has also been transferred into M Col 1505, a South American variety. CCMV-resistant TME 60444 transgenics are ready for field testing of the effectiveness of resistance as soon as biosafety regulations are in order in the relevant countries (N. Taylor and C. Fauquet, ILTAB/SCRIPPS, 1997, pers. com.). Transgenic cassava carrying genes for resistance to ACMV should follow about a year later. Transgenic approaches to bacterial blight resistance, globally the most widespread cassava disease, are in the experimental stages (Thro et al., 1998a in press).

Transgenic approaches to other concerns of cassava farmers. Concerns of cassava farmers centre most often around production level and stability, and market and price factors that relate to cassava root quality. Transgenic approaches will create new variation for root quality, especially through altered starch quantity and quality (Visser and Jacobsen, 1993). Genes for starch alteration (Salehuzzaman et al., 1992, 1993; Munyikwa et al., 1997) and modification of cyanogenic glycoside metabolism (Hughes et al., 1994) are now available for cassava. New opportunities for cassava through transgenesis research may come through:

• Reduced post harvest deterioration for better market access and reduced crop losses. Genetic transformation will permit elucidation of the biochemical pathway of this trait, information that will be useful both to transgenic and other approaches to this trait.

• Cyanogenesis. Transgenic approaches will permit a variation of types of cyanogen metabolism for optimal management. Farmers will be able to chose cyanogen (toxin)-free varieties for uses that require 100% consumer confidence, and cyanogenic (toxic) varieties for other uses, when production or plant protection advantages are more important.

• New products. Ultimately, transgenic approaches may permit the use of the cassava root's uniquely rapid photosynthetic mechanism (Black et al., 1993) for synthesis of compounds such as biodegradable plastics.

• Better basic understanding of cassava's unique metabolism.

Cassava's unusual biology confers both advantages (unparalleled robustness in harsh environments) and disadvantages (an inherent slow multiplication rate, and build-up of systemic pests and diseases). Slow multiplication rate is the number one bottleneck to diffusion of new varietal technologies for small farmers; systemic infections cause losses in production and diversity. In vitro techniques can overcome these disadvantages. Micropropagation, thermotherapy, and in-vitro slow growth already play essential parts in conservation and exchange of cassava diversity.

Rapid in-vitro varietal multiplication for cassava is in the pilot testing stage in Africa and South America. Robust, low-cost locally-sustainable methods are the objective. Another in-vitro technology, genetic transformation, is in the experimental stage.

To foster the desired social and economic benefits from rapid multiplication and transgenic technologies will require an integrated approach involving the combined knowledge of farmers, researchers, and processing/market experts. By creating linkages among these groups, CBN weaves together two threads: strategic development of in-vitro technologies for cassava, and their participatory development and evaluation with farmers and processors.

The authors wish to acknowledge the contributions of CBN's Steering Committee and Scientific Advisory Committee and of CBN's research members to the strategy and activities presented in this paper. DGIS/BIOTECH, the Government of the Netherlands Special Programme on Biotechnology and Development Cooperation, has made possible the development of the linkages described, through its support to CBN Coordination.

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