Ivan Ingelbrecht, i.ingelbrecht@cgiar.org

Cassava, photo by IITA

Cassava plays an important role in the food security of many developing nations, especially in Africa, and is also an industrial crop in Latin America and Southeast Asia. Native to South America, cassava was introduced to West Africa from Brazil by Portuguese traders in the 16th century. In the 1700s, cassava was independently introduced into East Africa, also by the Portuguese. Because of its high adaptability to low-fertility soils and its ability to withstand erratic and long periods of drought, cassava is a food security crop and a source of cash for resource-poor farmers. Today, over half of the world’s cassava is produced in Africa; Nigeria is the main producer.

As cassava is vegetatively propagated, genetic improvement is arduous because of the high levels of heterozygosity and poor flowering habit in many landraces. Genetic transformation could complement conventional breeding efforts for improving certain traits. Protocols for the genetic modification of model cassava genotypes, such as TMS60444, were first described in 1996 and have now become routine (Taylor et al. 2004).

Despite this progress, the genotypes that are amenable to genetic modification are not grown by farmers or used as genetic stocks in breeding programs, and this limitd their practical utility and impact at the farm level. Because the current transformation methodologies are highly genotype-dependent and limited to these so-called model genotypes, the genetic transformation of farmer-preferred cassava landraces remains a challenge.

State of cassava genetic transformation
Genetic transformation of cassava was first independently described in 1996 by three research groups. Nowadays, there are at least three modes of cassava genetic transformation, all of which rely on the establishment of somatic embryogenesis.

Expression of a reporter gene in leaf, root, and stem of the cultivar Tokunbo, a transgenic African cassava. Photos by IITA

In one, friable embryogenic callus cultures (FEC) are used to transform and regenerate plantlets. This technology works well with TMS60444 and a few South American varieties, such as MCol 1505. This protocol can yield large numbers of transformants and is possibly the most widely used technology to date. Using this method, several new traits have been introduced into cassava. A major international effort involving research organizations in the USA, Europe, Asia, Latin America, and Africa, aims at enhancing the nutrition of cassava through the genetic modification of TMS60444. Although of African origin, this variety is not used by farmers or breeders as it has unfavorable disease and agronomic characteristics. However, over the past decade, this transformation methodology has provided proof of the concept with genes in TMS60444.

A second procedure relies on transformation of embryogenic cells present on immature leaf lobes. Because this technology is faster, it may result in less somaclonal variation than the FEC system since the somatic embryos are not derived from long-term cultures. This methodology also has limitations in terms of farmer-preferred genotypes.

A third methodology uses somatic embryo explants that undergo shoot regeneration (organogenesis). This procedure was first developed by Li et al. (1996) for model cassava genotypes and has been further enhanced by IITA and its collaborator, the University of Copenhagen KU), Denmark, for the African farmer-preferred landraces.

At IITA, somatic embryogenesis has been established for four cultivars: one from West Africa and three from East Africa. These varieties are susceptible to cassava brown streak disease (CBSD) but otherwise have desirable agronomic characteristics. For these four varieties, thousands of somatic embryos (SEs) can be reproducibly generated. Using somatic embryos as source explants, transgenic Tokunbo, a landrace cultivated in several countries in West Africa, has been produced using a beta-glucuronidase (GUS) gene construct.

Transgenic cassava Tokunbo in the greenhouse. Photo by IITA

Enabling technologies
In addition to genetic transformation protocols, a set of other technologies is required for biotechnology-mediated cassava improvement. Since many traits target specific tissues, such as roots, a wider range of promoters or other regulatory elements needs to be investigated and tested in cassava to achieve optimal and stable transgene expression in these tissues or in the whole plant. The aspect of stable gene expression, i.e., the absence of gene silencing, is especially relevant for a vegetatively propagated crop, such as cassava. The international effort to sequence the cassava genome as well as initiatives of several labs, including IITA, to develop functional genomics tools will greatly expand our tool set for biotechnology-mediated cassava improvement, whether through transgenesis or marker-assisted breeding approaches.

Many biotechnologies are protected by intellectual property rights and thus are not always freely accessible, especially to research institutions in the developing world. In this context, a new plasmid was developed at IITA for Agrobacterium-mediated genetic transformation of cassava and other dicotyledonous plants. This plasmid contains a Cassava Vein Mosaic Virus promoter cassette and was tested in tobacco and cassava using a beta-glucuronidase reporter gene. High, constitutive expression was obtained in leaf, stem, and root tissues of both crops, thus showing that the new promoter cassette was fully functional.

A derivative of this plasmid was made by IITA’s partner, the German Resource Centre for Biological Materials (DSMZ) to manipulate gene expression via RNA interference. RNA interference is widely used in animal and plant cells to down-regulate gene expression. In cassava, this approach is currently being used to engineer virus-resistant plants and plants that produce reduced levels of cyanide.

Preparing cassava somatic embryo tissues for shoot induction. Photo by IITA

Beyond genetic transformation
One of the highlights at the International Congress of Genetics, held in Berlin in 2008, was a report on the use of artificial mini-chromosomes in maize. The methodology makes use of artificial mini-chromosomes that can replicate and are stably transmitted over several generations, in a way similar to natural chromosomes. These mini-chromosomes contain DNA sequences found in centromeres, the chromosomal regions needed for inheritance. Rather than inserting new genes randomly into a plant’s natural chromosomes, as happens in genetic modification, these mini-chromosomes remain separate. One of the advantages of this technique is that it allows multiple genes to be arranged in a defined sequence with their own regulatory sequences, resulting in more consistent gene expression. So far, this technique has been used only in maize but it can potentially be applied to other crops as well, including cassava.

Outlook
Although methods for cassava genetic transformation were developed over a decade ago, genetic transformation of farmer-preferred (African) cultivars has only recently become a research priority. IITA and its partner KU in Denmark have recently produced a first transgenic cassava variety called Tokunbo which is cultivated in Nigeria and some other West African countries.

Cassava plant. Photo by IITA

As a complementary approach to cultivar-specific transformation, the movement of transgenes through conventional breeding should also be considered and, ideally, also artificial mini-chromosomes, the new gene-transfer technology established in maize.

As new and cost-effective DNA sequencing methodologies are becoming mainstream, the amount of genomics and genetic information for cassava will vastly increase in the near future and greatly expand our tool set for biotechnology-mediated improvement. From a technical standpoint, it is realistic to expect that new varieties, in part produced using biotechnology tools, will become available in the near future.

References
Li H-Q, C. Sautter, I. Potrykus, and J. Puonit-Kaerlas. 1996. Genetic transformation of cassava. Nature Biotechnology. 14: 736–738.

Taylor N., P. Chavarriaga, K. Raemakers, D. Siritungaand, and P. Zhang. 2004. Development and application of transgenic technologies in cassava. Plant Molecular Biology 56: 671–688.