Mark Halsey

Isolation of Maize from Pollen-Mediated Gene Flow by Time and Distance

Development of improved genetic traits in maize (Zea mays L.) requires robust measures to prevent pollen-mediated gene flow (PMGF) and assure isolation of new traits whether these traits are the result of conventional breeding or of modern genetic techniques. Studies were conducted in California and Washington to evaluate the relationship of distance and temporal separation for isolation from PMGF. Kernel color was used to detect outcrossing from source plots of 0.4 to 1.2 ha in size to receptor plots planted at distances up to 750 m and planting intervals of up to 3 wk from the pollen source. Outcrossing from source to receptor plots was observable to 0.0002% (1 kernel in 500 000 kernels). Increasing temporal separation reduced the distance required to achieve genetic isolation. Outcrossing was <0.01% at 500 m when source and receptors flowered at the same time, whereas this level of confinement was achieved at 62 m or less when 2 wk of temporal separation was used. No outcrossing was detected at 750 m and 2 wk of temporal separation. This is the first practical evaluation of time and distance acting together to achieve genetic purity in maize.

Transgenic RNA interference (RNAi)‐derived field resistance to cassava brown streak disease

Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (ΔCP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length ΔCP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718-005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Cross-protection against CBSV by siRNAs generated from the full-length UCBSV ΔCP confirms a previous report in tobacco. The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease.

The VIRCA Project: virus resistant cassava for Africa.

The VIRCA (Virus Resistant Cassava for Africa) project is a collaborative program between the Donald Danforth Plant Science Center, USA the National Crops Resources Research Institute, Uganda and the Kenya Agricultural Research Institute, Kenya. VIRCA is structured to include all aspects of the intellectual property, technology, regulatory, biosafety, quality control, communication and distribution components required for a GM crop development and delivery process. VIRCAs goal is to improve cassava for resistance to the viral diseases cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) using pathogen-derived RNAi technology, and to field test, obtain regulatory approval for and deliver these products to small landholder farmers. During Phase I of the project, proof of concept was achieved by production and testing of virus resistant plants under greenhouse and confined field trials in East Africa. In VIRCA Phase II, two farmer-preferred varieties will be modified for resistance to CBSD and CMD, and lead events identified after molecular and field screening. In addition to delivery of royalty-free improved planting materials for farmers, VIRCA capacity building activities are enhancing indigenous capability for crop biotechnology in East Africa.

Strategic environmental assessments for genetically modified organisms

Genetically modified crops appear to provide a promising option in finding sustainable solutions to end global hunger and poverty, but strategic decisions need to be made on how to spend limited agricultural research funds. Potentially, strategic environmental assessment (SEA) may be used as part of an environmental management system to introduce mainstreaming of environmental considerations in the policy research and priority-setting process of development organizations to help achieve international development goals. This paper sets out a possible biotechnology SEA process that integrates qualitative and quantitative assessments with a focus on risk assessment and management within the SEA and policy environmental assessment frameworks. It uses the International Association for Impact Assessment six performance criteria for SEAs: integration; sustainability; focus; accountability; participation; and iteration.

Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations

Cassava brown streak disease (CBSD) presents a serious threat to cassava production in East and Central Africa. Currently, no cultivars with high levels of resistance to CBSD are available to farmers. Transgenic RNAi technology was employed to combat CBSD by fusing coat protein (CP) sequences from Ugandan cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) to create an inverted repeat construct (p5001) driven by the constitutive Cassava vein mosaic virus promoter. Twenty-five plant lines of cultivar TME 204 expressing varying levels of small interfering RNAs (siRNAs) were established in confined field trials (CFTs) in Uganda and Kenya. Within an initial CFT at Namulonge, Uganda, non-transgenic TME 204 plants developed foliar and storage root CBSD incidences at 96–100% by 12 months after planting. In contrast, 16 of the 25 p5001 transgenic lines showed no foliar symptoms and had less than 8% of their storage roots symptomatic for CBSD. A direct positive correlation was seen between levels of resistance to CBSD and expression of transgenic CP-derived siRNAs. A subsequent CFT was established at Namulonge using stem cuttings from the initial trial. All transgenic lines established remained asymptomatic for CBSD, while 98% of the non-transgenic TME 204 stake-derived plants developed storage roots symptomatic for CBSD. Similarly, very high levels of resistance to CBSD were demonstrated by TME 204 p5001 RNAi lines grown within a CFT over a full cropping cycle at Mtwapa, coastal Kenya. Sequence analysis of CBSD causal viruses present at the trial sites showed that the transgenic lines were exposed to both CBSV and UCBSV, and that the sequenced isolates shared >90% CP identity with transgenic CP sequences expressed by the p5001 inverted repeat expression cassette. These results demonstrate very high levels of field resistance to CBSD conferred by the p5001 RNAi construct at diverse agro-ecological locations, and across the vegetative cropping cycle.