Principles of Plant Genetics and Breeding

380 CHAPTER 20
Initial proof of concept for the ability of transgenically expressed AC1 to impart resistance to the geminiviruses causing CMD
was achieved using tobacco plants. The production of transgenic tobacco (Nicotiana benthamiana) is straightforward and inexpensive.
In comparison, recovering transgenic cassava is labor intensive, requires specially trained staff, and requires 5–6 months
to recover plantlets. N. benthamiana is susceptible to infection with a range of virus species, making it a highly effective model
species for such studies. Research by workers at the DDPSC and in the UK demonstrated that, indeed, when the AC1 gene was
integrated into tobacco, the resulting plants were significantly more resistant to infection by cassava geminiviruses than the nontransgenic
control plants (Hong & Stanley 1996; Sangaré et al. 1999). Armed with this information, workers could progress with
some confidence to the next stage and develop this strategy in cassava itself. Technologies described above were utilized to
regenerate approximately 20 transgenic cassava plants from independent integration events in the CMD-susceptible, West
African variety 60444. Presence of the transgenes was confirmed by polymerase chain reaction (PCR) amplification for both the
AC1 and the nptII selectable marker genes in the FEC lines and subsequently in regenerated plantlets.
Production of transgenic plants containing a desired transgene does not guarantee that the desired trait will be expressed at the
level needed to generate a product of the quality required for release to farmers. In order to test for efficacy of the AC1 gene in cassava,
transgenic cassava plants were challenged with infectious clones of three geminivirus species. Although whiteflies are the
natural vector for CMD, maintaining virifilous populations of the specific biotype responsible for spreading the disease in Africa is
highly problematic in the temperate regions. As an alternative, viral DNA was coated onto micon-sized gold particles and shot
into young cassava plants using Biolistic® microparticle bombardment technology. When cassava plants were inoculated in this
manner, four lines were found to exhibit significantly enhanced resistance to the pathogens (Chellappan et al. 2004). Most interestingly,
these plants were resistant not only to African cassava mosaic virus (ACMV) but also to East African cassava mosaic virus
(EACMV), Sri Lankan cassava mosaic virus, and to simultaneous, dual challenge with ACMV and EACMV.
Cross-protection imparted by the AC1 transgene from ACMV was not predicted at the beginning of the project, but has important
implications for deploying such a technology to the field in Africa. As described above, there are at least six species of geminiviruses
infecting cassava in sub-Saharan Africa. An effective defense strategy against CMD, whether developed through
conventional breeding or biotechnology, must provide protection against most or all of these, and also against the synergistic
effects of dual infection with more than one species of viral pathogen. Small interfering RNAs (siRNAs) specific to the AC1 gene
were detected in the two plant lines showing highest resistance to CMD prior to infection with virus (Chellappan et al. 2004), indicating
that the AC1 transgene was triggering gene silencing mechanisms against this sequence within the plant. The AC1 gene is
well conserved across different species of cassava-infecting geminiviruses, and it is therefore hypothesized that such siRNAs are
capable of targeting the mRNA of this sequence for degradation immediately the transgenic plant becomes infected with a geminivirus,
thereby inhibiting viral replication and imparting resistance.
To summarize the above it can be stated that by integrating one transgene (two if the nptII selectable marker gene is counted)
we were able to generate very resistant plant lines from an otherwise highly CMD-susceptible cassava cultivar. The caveat to this
proof of efficacy was that resistance was demonstrated in the greenhouse in the US midwest and plants were inoculated with the
pathogens in an artificial manner. Important questions remain as to whether the plants will perform well when grown under field
conditions in Africa where the viruses are transmitted by whiteflies. Testing the plants under such conditions is a critically important
step in demonstrating the usefulness of this technology. Success at this stage is critical to deciding whether to proceed along
the delivery pathway.
Establishing field trials of transgenic plants is straightforward in North America where many thousands of such tests have been
completed over the last decade. The situation in Africa is dramatically different. As of late 2004, outside of South Africa, only
Kenya, Zimbabwe, and Burkina Faso have carried out field trials, with the total combined number being less than 10 trials. The
remaining countries either do not have the required regulations, biosafety infrastructure, and trained personnel in place, or have
never exercised their existing regulatory procedures to establish field trails of transgenic plants. This situation obviously presents
challenges to those wishing to develop and deploy a transgenically enhanced crop in such regions. The DDPSC has established
collaboration with the Kenyan Agricultural Research Institute (KARI) to carry out trials of the cassava plants described above. After
consideration by the Kenyan National Biosafety Committee it was recommended that a contained screenhouse trial should be
performed in a cassava-growing region of western Kenya – the first test of transgenic cassava in Africa. Plantlets were exported
from the DDPSC to Kenya and planted in pots in a biosafety level 2 screenhouse constructed for the trial. At the time of writing,
whitefly inoculation of transgenic and control plants is ongoing within this structure. If the transgenic plants demonstrate acceptable
levels of resistance to the geminiviruses infecting cassava in western Kenya, it is planned to carry out multilocational,
confined field trials in this and other regions of that country.
The product development and delivery pathway contains a loop back from the field trial phase to the laboratory. There are two
reasons as to why this takes place. Firstly, the technology in question could perform well in the field, but not well enough to proceed
in its initial form to further product development and delivery. In this case more work is required in the laboratory, most
likely to improve transgene express and/or to direct this expression to desired tissues in a more effective manner. Further testing in
the greenhouse and field will then be required to confirm any improvements. For crops such as cassava, even if initial field trials
are successful, it will be necessary to return to the laboratory in order to generate more genetically transformed plants. This is
because the variety in which the initial transgene integrations take place is most likely not in the background most suitable to
farmers’ needs. In sexually propagated crops it is possible to take the best performing transgenic plant produced and backcross
this with preferred parents within established breeding programs. In this way the beneficial transgenic trait is introgressed into a