Full and durable crop resistance in rice and potato plants to nematodes. Final Technical Report.
Abstract
The aim is to deploy plant genes expressed in food as transgenes to achieve full and durable resistance to nematodes with expression limited to roots systems. The work centred on a monocot (rice) and a dicot plant (potato). The approach has potential against a range of nematodes but the work has emphasised Meloidogyne. DFID and others have identified this genus as the most important nematode problem of developing world agriculture. The intention is to retain cultivars that are preferred by local populations and not change agronomic practices. The plants will also be used to champion the benefits of transgenic crops for poor growers. The work on rice is collaboration with John Innes Centre in which they use the constructs we make to transform rice for us to evaluate. Constructs were made for biolistic transformation and to replace that approach with one that is Agrobacteriummediated. On-going development of the latter approach at JIC to a variant that avoids selectable markers has resulted in R7294 necessarily concentrating on biolistically-transformed rice. Rice expressing a cystatin from maize included lines (NE lines) that provided resistance levels of 83 ± 6% and so effectively met the level of resistance targeted at the start of the work. These plants grew normally. Co-transformation with the same construct and one expressing Chicken egg-white cystatin did not raise resistance further. Possibly a higher level or resistance will not be achieved by expressing just cystatins additively. Analysis of transcripts from lines generated in previous work confirmed that silencing occurred in some biolistically transformed lines. However an homozygous line were identified in T2 seed of the NE line. These plants supported little reproduction of Meloidogyne and expressed cystatin. T3 of this line was generated. Sufficient seed of T3 is already available for small-scale field trial and a visit is planned to China in May 2002 to set up these trials and parallel effort for potato. The objective is determine efficacy and establish if reliable field trial conduct and analysis with these plants. Once the selectable marker-free rice plants expressing cystatin are generated, they will replace current rice plants in field evaluation. The activity of promoters of a root specific tubulin (TUB-1) was studied in rice. It showed very little activity green tissues and was not very active in older root tissues. However it was highly active in the giant cells induced by M. incognita on rice. This enables a cystatin to be targeted to the feeding nematode with little activity elsewhere in the plant. Therefore the protein will not be present in rice used as food. This adds considerably to the inherent biosafety given that maize cystatin occurs in food and is neither a human toxin nor allergen. An effort parallel to that on rice was completed for potato. A number of constructs we made and three field trials were carried out in the UK against Globodera which is an important pest in Bolivia. A Bolivian government temporary moratorium prevented transgenic trial in that country. Trials established that plant cystatins are as effective as that from chicken egg white which has been used as a standard for comparison. The second trial showed that the three root preferential provided as high a level of resistance as when a constitutive promoter controlled expression. Additive resistance was established using a cystatin to improve the partial natural resistance of both an Andean (Maria Huanca) and an UK cutlivar (Sante) to full resistance in a containment and UK field trial respectively. This establishes a paradigm for additive resistance using different genes that act distinctly against nematodes. Further additive constructs were made and the first field trialled in 2001 but analysis is not yet complete. The transgenic Desiree plants that showed resistance to Globodera were also resistant to M. incognita in containment. These plants did not show reduced growth in either the field or containment. Bolivian S. tuberosum andigena cultivars were transformed but their expression levels were lower than obtained for the hybrid Maria Huanca. Promoter reporter constructs were made, expressed and studied in potato. Results were similar to those recorded for rice. The three promoters showed little or no activity in green tissues but all were active in the giant cells induced by Meloidogyne. This effect was confirmed with one promoter within the syncytium induced by G. pallida. Therefore promoter/cystatin constructs could be deployed within pest management strategies to provide a range of nematode resistant crops for subsistence farmers. The perceived hazard of transgenic potato release into centres of biodiversity for solanaceae was addressed. Male sterility was achieved by delivering an RNAase or a ribosomal inactivating protein from maize seed under control of a promoter specific to male parts of the flower (tapetal specific promoter). Also distinctive forms of potato were established for future transformation based on both flower colour and leaf shape. Such plants would support farmer choice to grow or avoid GM plants. Phenotypic markers are important where literacy levels ensure written labels are inadequate. Such plants would also allow socioeconomists to measure uptake by growers in the future.
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