CIAT has several breeding strategies for beans, Brachiaria grasses, cassava, and rice. One component comprises genetic transformation technologies, which are tested in key cases where standard alternatives are much less
(A) Symptoms of RHBV; (B) Vector RHBV.

promising because of a lack of genes, a need to modulate the expression of existing traits, or new traits are being introduced.

The traditional focus of genetic transformation was to introduce novel sources of resistance to biotic stresses. Currently, more attention is being given to food and feed quality traits and plant environmental adaptation. Food and environmental biosafety issues are also taken into account in the development of projects. Environmental biosafety research in the area of analyses of gene flow into wild or weedy relatives in the tropics is being integrated as a component in the benefit-to-cost analyses of this technology.

Common Bean

A methodology for Agrobacterium-mediated genetic transformation of mature seed meristems was developed for hybrids of cultivated and wild genotypes of tepary bean (Phaseolus acutifolius). The common bean (Phaseolus vulgaris) has proven highly recalcitrant for this transformation methodology. Our current efforts focus on the use of a double congruity backcross scheme to introgress higher regeneration and transformation response from P. acutifolius into P. vulgaris while preserving desirable agronomic traits of common bean. Transgenic hybrids are being generated using this approach. Also, the methodology for genetic transformation of common bean elite genotypes through particle bombardment is being optimized. Traits of interest include drought tolerance. 

Brachiaria Grasses

We developed a protocol for recovering transgenic plants from direct gene transfer and filed for intellectual protection, jointly with EMBRAPA, in both Brazil and Colombia in 2000. We are developing Agrobacterium-mediated transformation so we can use methods for delivering cleaner transgenic events. We are also targeting molecular breeding for Brachiaria to overcome limitations in forage quality, such as dry matter indigestibility and water-insoluble carbohydrates. We have begun work on cloning key genes involved in lignin biosynthesis.

Cassava

We are using Agrobacterium tumefaciens to genetically transform somatic embryos (i.e., friable embryogenic callus or FEC) of cassava clones used by small farmers. The research agenda is broadening to include added-value traits in cassava varieties for industrial uses, such as starch and animal feed. Herbicide-resistant cassava was the first product developed, thus permitting the use of minimum tillage practices for soil conservation. New lines from farmer preferred cultivars are being produced in 2003.

We are also introducing into cassava resistance to the stemborer Chilomina clarkei. This major pest was first reported in the 1990s as affecting poor farmers of the Atlantic coastal regions of Colombia and Venezuela. It is now spreading throughout Colombia, Bolivia, and northern Argentina. So far, no genetic resistance has been reported for this pest. Transgenic strategy includes introducing insecticidal proteins such as cry1 Ab. Transgenic cassava carrying the gene for this protein is being evaluated for its efficacy in controlling this borer. Low levels of protection have been observed, although further experiments are needed to confirm these findings.

Work is progressing on cassava with novelty starch qualities, and on cassava with increased contents of b-carotene (precursor of vitamin A) in its roots.

Rice

Transgenic rice has provided a learning experience in scaling up the use of transgenesis in breeding at CIAT. Efficient reproducible methods, using either particle bombardment or Agrobacterium-mediated genetic transformation, have been optimized to transform recalcitrant indica rice varieties adapted to flooded conditions. The technology is also applied to genotypes adapted to acid soils or high-altitude Andean ecosystems.

The most advanced project is the generation of plants resistant to the rice "hoja blanca" virus (RHBV), a major endemic viral disease of economic importance in tropical America. The virus's genome was characterized at CIAT. Viral genes were cloned and introduced into 'CICA 8', the rice variety most widely grown by small farmers and heavily used in breeding as a parent. The best transgenic lines outperformed commercial varieties in the field. Advanced crosses generated with this transgenic resistance source are being bred to deploy the resistance into a broader range of materials.

Current work involves developing rice with resistance to sheath blight, for which field tolerance, but not genetic resistance, is available. Future work includes plant environmental adaptation and quality traits.

Genetic Transformation and Breeding

Scaling-up operations to introduce transgenic lines into breeding have been implemented gradually. An interdisciplinary approach is followed to evaluate transgenic plants in the greenhouse and field.

Precision Genetic Engineering

High priority is given to public acceptability. New emphasis is being placed on precision genetic-engineering technologies that allow the development of clean transgenic events that express the gene of interest in the targeted tissue without having to use antibiotic or herbicide-resistance selection marker genes. CIAT employs genes from plants or related organisms.

Environmental Biosafety

CIAT is actively engaged in assessing the potential impact, in the tropics, of gene flow from transgenic plants entering the genetic structure of populations of their wild or weedy relatives. Models include native species of crops, farmer field experimental sites, and other factors such as landraces, thereby giving a comprehensive scenario applicable to the tropics worldwide.

The type of products expected from biosafety research is governed by the need to efficiently develop low-input varieties with an increased quality that would meet the needs of national partners. An interdisciplinary approach is being used toward biosafety in the expectation that expertise on molecular and population genetics, agroecology, wild habitat ecology, and socioeconomic analyses would provide an improved assessment of the costs and benefits of this technology.

CIAT also actively participates in capacity building of national partners in biosafety. For the last 3 years, CIAT developed courses for the National Biosafety Technical Council of Colombia, breeders from Latin America and the Caribbean, and journalists.

Future Plans

• As soon as suitable alternative technologies are available, to cease using selectable markers and introduce only the gene or genes of interest into the final product
• To fully implement a system that will allow the expeditious analysis of efficacy of a transgene or transgenes in plants, and breed accordingly, taking into account the corresponding biosafety measures
• Introduce new traits for increasing both environmental adaptation to marginal agricultural conditions and nutritional quality of food and feed
• Contribute to CIAT's biosafety research on non-targeted organisms associated with plants or soil biota
• Strengthen national partners' training and participation in biosafety research.

The Breeding-genetic Transformation Team

Paul Chavarriaga, Alvaro Mejía, Hernán Ceballos, César Martínez, John Miles, Tony Bellotti, Fernando Correa, Lee Calvert, Martin Fregene, Matthew Blair, Joe Tohme, Myriam Duque, Daniel Debouck, Zaida Lentini