Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Establishment of homozygous lines of transgenic rice expressing GNA lectin, PHA-E lectin and Bt toxin, respectively

The specific objectives were to produce transgenic rice plants expressing snowdrop lectin (GNA), Phaseolus vulgaris agglutinin E-form (PHA-E) and Bacillus thuringiensis toxin (Bt) constitutively expressed throughout the rice tissue and therefore provide the necessary transgenic plant material for the programme. Quantitative immunological techniques were employed to assay levels of accumulation of transgene products in the transgenic rice plants, and to generate lines of progeny plants containing defined levels of transgene products in the harvested seeds.

Primary transformants of rice plants containing the gene constructs for expression of the selected foreign proteins (GNA, PHA-E) were produced by particle gun bombardment of mature, seed-derived callus. Calli were bombarded with gold particles coated with DNA of the plasmids to be co-transformed together with the plasmid containing the selectable marker gene. After bombardment, calli were transferred to proliferation medium to allow plantlet formation. Plantlets were separated, transferred to rooting medium and after root induction, to soil. Plantlets were screened for transgene presence by PCR while still in tissue culture (when approx. 5cm high), using primers directed against the appropriate transgene coding sequence. Plantlets negative for the transgene were discarded at this stage. Rooted plantlets growing on in soil were screened for the presence and accumulation of the transgene product by immunological assay (Western blotting after analysis of total protein by SDS-PAGE). The remaining plants were grown on to maturity in growth rooms under high-light conditions, and allowed to set seed by selfing.

Immuno-dot blot assays were used to quantify levels of accumulation of transgene products in selected primary transformant rice plants; quantification was carried out by densitometry of X-ray films exposed to the blots, using Bio Rad Molecular Analyst software. Based on these assays, positive and negative expressors for the transgenes were selected. These plants were used as the basis for transgenic rice lines and controls. Seed from subsequent generations were assayed to establish defined levels of transgene product accumulation in the selected lines.

PHA-E expression at the mature stage in the selected lines was highest in seeds (approximately 0.78% total soluble protein), and lowest in the root (Approximately 0.051% total soluble protein). Stability of transgene expression was also assessed employing quantitative immunoassays (dot-blot) in progeny plants from the selected primary transformants. The expression levels in seeds from three successive generations (T1-T3) was 0.75, 0.74, and 0.77% of the total soluble protein respectively, indicating no significant differences over time. At T4 (the latest generation) seeds still showed some degree of heterozygocity.

GNA expression levels were highest in seeds (0.98%) and lowest in leaves (0.25%). As with PHA-E, GNA expression in the seeds was relatively constant over three consequetive generations (T1-T3, 1.01, 1.4, 1.2% respectively). Seeds from the final generation (T6) were all homozygous after screening by immunoassay.

Bt toxin expression levels were lowest in seeds (0.018%) and highest in the leaf sheath (0.28%). The level of expression in seeds over three successive generations (T9-T11) was 0.017, 0.016, and 0.015% respectively, showing no significant differences over time. The final generation of the seeds (T11) were homozygous.

More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

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University of Newcastle
University of Newcastle upon Tyne
NE1 7RU Newcastle upon Tyne
United Kingdom
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