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Engineering rice for resistance to insects (ERRI)

Contract nr: FAIR-CT97-3761
Project nr: 3761
Project type: SC
Starting date: 01/03/1998
Duration: 48 months
Total cost: 1,520,714 EUR
EC Contribution: 1,060,000 EUR
Scientific Officer: Richard HARDWICK
Research topic: Plant health
Acronym: ERRI

The striped stemborer (SSB) (Chilo suppressalis) is one of the major constraints on rice production in southern European countries, causing yield losses reaching 15 to 20% in Spain and France. To address this problem, heavy chemical treatments as well as sprays with Bacillus thuringiensis insecticidal formulations have proven of limited efficiency, due to their poor persistence and to the biological localisation of the pests, which develop in rice culm. Non-selective chemical spraying is environmentally damaging. Conventional breeding for that trait has proven to be difficult.

The goal of this project is to develop SSB-resistant rice through transfer of Bacillus thuringiensis (B.t.) genes encoding insecticidal toxins and plant proteinase inhibitor (p.i.) genes into the two most widely used rice varieties in Italy, Spain and France.

This project comprises:
1) the use and synthesis of sequence-optimised B.t. toxin genes encoding proteins previously identified as specifically active against SSB larvae by affinity and toxicity tests;
2) the use of maize and soybean protease inhibitor genes;
3) the construction of vectors for expression of the transferred genes in rice plants, including constitutive promoters conferring high-level expression to foreign genes in transgenic rice plants, a vascular tissue-specific promoter of the maize streak virus and wound-inducible promoters of maize and tobacco genes;
4) the use of transformation systems optimised for two Mediterranean elite varieties based on coculture of embryogenic calluses with Agrobacterium tumefaciens;
5) the production and bio-assay for SSB resistance of transgenic plants containing: a single B.t. gene; a combination of two B.t. genes under the control of a constitutive promoter; one or two B.t. genes under the control of promoters having tissue specific or wound inducible activity; one p.i. gene; two p.i. genes exhibiting complementary specificities for insect proteinases;
6) the proposition of strategies for deploying the selected transgenic lines in order to delay and hopefully prevent occurrence of resistant forms of SSB;
7) the assessment of the gene-flow rate between transgenic rice and its derived weed form, the red rice.
The deployment of SSB-resistant, elite, Mediterranean rice varieties generated in this project will eventually result in a significant decrease of pesticide use in fragile, rice-growing areas of south Europe.

Current situation/results:
Full synthesis of the cry1Aa and cytA genes and functional analyses of tissue specific and wound-inducible promoters have been completed. Agrobacterium vectors bearing the cry1Aa or cry1B gene placed under the control of either the constitutive maize ubiquitin or the wound-inducible hrgp or mpi promoters have been used to transform Ariete and Senia varieties. Early generated microprojectile bombardment-mediated transformation events of the two cultivars harbouring the cry1B gene or a soybean or maize proteinase inhibitor gene have been characterised at the molecular level and have been or are being challenged with SSB larvae. Full protection was attained from T0 to T4 generation in Ariete and Senia events transformed with the ubi-cry1B or mpi-cry1B construct. Accumulation of Cry1B in leaf and pith tissue upon wounding and absence of Cry1B in pollen and grain endosperm was demonstrated in mpi-cry1B plants. Field trials designed for assessing gene flow between transgenic and conventional rice have been successfully conducted in Italy and Spain. Resistance acquirement studies with laboratory rearings of SSB larvae fed on artificial diet containing LC50 doses of Cry toxins have reached the seventh generation without increase of resistance.


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