Principles of Plant Genetics and Breeding

BREEDING FOR RESISTANCE TO DISEASES AND INSECT PESTS 379
to be recovered. In the first, it was discovered that when the organized embryogenic structures (OES) were transferred from a
Murashige and Skoog-based medium to one containing the basal salts of Greshoff and Doy, the OES would break down into a
highly friable embryogenic callus (FEC) from which somatic embryos could be regenerated and plants recovered upon subculture in
a medium devoid of auxin (Taylor et al. 1996). This FEC is composed of tens of thousands of submillimeter-sized pre-embryogenic
units that proliferate via disorganized divisions from single cells at their surface. As such they act as excellent target tissue for
transgene insertion and recovery of genetically transformed plants, by both microparticle bombardment (Schöpke et al. 1996)
and Agrobacterium (Gonzalez-de Schöpke et al. 1996). In the second system, it was discovered that if cotyledons were allowed to
develop from the OES, shoots could be induced to develop via an organogenic process from the cut surface of these organs in the
presence of an appropriate cytokinin. This system resulted in the recovery of transgenic cassava plants after co-culture of the
cotyledons with Agrobacterium (Li et al. 1996). Subsequent research has also proven that it is possible to recover transgenic cassava
plants directly from leaf explants via Agrobacterium-mediated gene transfer (Siritunga & Sayre 2003). For a more detailed
description of genetic transformation systems in cassava, the reader is referred to a recent review by Taylor et al. (2004).
The production of transgenic cassava plants is now routine in five laboratories around the world. Based in Europe, the USA,
and at the Centro Internacional de Agricultura Tropical (CIAT) in Colombia, four of the five utilize the FEC system as the target tissue
of choice and all now employ Agrobacterium-mediated gene insertion for the production of transgenic cassava plants. As for
other crops, it has been found that in cassava, Agrobacterium more reliably generates plants with single copy insertions of the
transgene – an issue considered important with regard to the stability of transgene expression and future deregulation of genetically
modified plants for release to farmers. The traits presently being targeted within cassava transgenic programs reflect the
interests of the specific laboratories and include: resistance to virus disease, herbicide tolerance, increased resistance to insect
pests, and enhanced starch quality, elevated protein, and reduced cyanogenic content of the storage roots (Taylor et al. 2004).
Steps to produce a transgenic crop plant in the tropics using virus-resistant cassava as an example
As an example of the steps and processes involved in producing a transgenic crop plant for delivery to farmers in the tropics, we
will examine in more detail the program and strategies employed at the Donald Danforth Plant Science Center (DDPSC) to produce
cassava with resistance to cassava mosaic disease (CMD). CMD impacts cassava production throughout sub-Saharan Africa
and the Indian subcontinent and is caused by at least eight species of whitefly-transmitted geminiviruses. The geminiviruses that
infect cassava are single-stranded DNA viruses possessing two circular genomic components, each approximately 2.6 kb in size.
The disease rarely kills infected plants but can significantly reduce the production of storage roots in susceptible varieties. The disease
is most severe when plants are simultaneously infected with more than one species of geminivirus, when the two pathogens
act synergistically to overcome the plant’s defense mechanisms. It is estimated that CMD is responsible for 30–40% yield reduction
in Africa, which is equivalent to as much as 25 million metric tons of food each year. Workers at the DDPSC are employing
transgenic technologies to impart resistance to CMD in varieties already preferred by farmers and consumers in Africa.
Transgenic programs start with an idea based on existing knowledge about how integration and expression of a specific gene or
genes might impart beneficial agronomic characteristics within the crop of interest. Before commencing work in the laboratory, it is
essential to determine whether the strategy in question can pass the rigorous food and environmental safety testing that all transgenic
plants must satisfy before they can be grown by farmers. For example, a gene that codes for a toxin from scorpions, or a known human
allergen, might provide very effective resistance against a given insect pest when expressed transgenically in a crop plant. However,
it can be predicted that such a product will not be acceptable to the regulatory agencies (nor the public at large) and can therefore
never be made available to farmers. Proceeding with such a program may be justified on a purely scientific basis, but if the intention
is to bring a new product to market, it would be a waste of resources and the program would be terminated before it began.
Likewise, before significant investment is made in the research and development phases, scientists must work with lawyers
experienced in the field of intellectual property rights. Many genes, genetic sequences, transformation protocols, and other tools
required to develop a given transgenic plant are protected through patents and licensing agreements. Failure to obtain the “freedom
to operate” for commercial release early in the development program for all the technologies needed to develop the final
product, can cause significant problems later in the product delivery process. It is at that time that the owners of such technologies
may assert their legal rights and demand royalties or otherwise block commercial release. A well-known example of such problems
arose with “Golden Rice”, where unresolved intellectual property right issues were not addressed until late in the development
process. Significant resources were required to resolve outstanding issues and further product development was delayed.
Workers at the DDPSC have adopted a pathogen-derived resistance strategy to combat the effects of CMD. In such approaches,
genetic sequences from the viral pathogen are cloned, fused to an appropriate expression cassette, and integrated into the plant’s
genome. In this specific case the AC1 (or replication associated) gene from African cassava mosaic virus (a causal agent of CMD)
was chosen as the candidate gene. AC1 and the use of replication-associated genes to impart resistance to geminiviruses resides
in the public domain and therefore its use is not restricted due to issues of intellectual property. The promoter sequence used to
drive expression of this gene in cassava was the cassava vein mosaic virus (CsVMV) promoter, previously isolated and characterized
at the International Laboratory for Tropical Agricultural Biotechnology (ILTAB) (Verdaguer et al. 1998). Although licensed to a
commercial entity, it is available through special agreement for use and release within transgenic products to farmers in developing
countries. A history of safe use for pathogen-derived resistance technologies deployed to farmers in transgenic papaya, potato,
and squash, generated confidence that this approach in cassava would not encounter problems of food and environmental safety,
should a successful product be developed.