By D. Valvekens, A. Van Gysel, M. Van Montagu, M. Van Lijsebettens (auth.), Professor Dr. Ingo Potrykus, P.D. Dr. German Spangenberg (eds.)
Using the in actual fact dependent protocols given during this handbook, will probably be effortless to use the newest concepts in plant biotechnology which will create new plant types or types with altered and optimized characteristics.
Direct gene move into plant cells or protoplasts by way of microinjection, electroporation or biolistic structures, or mediated by way of Agrobacteria is defined intimately for varied plant species, together with appropriate vegetation and cereals. additionally incorporated are protocols of the traditional molecular thoughts for the research of transgenic vegetation in addition to a piece on biosafety issues and regulations.
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The youngest leaves at the apical meristem and the oldest leaves at the base of the plants will not give rise to transformed calli or shoots at a good frequency. A second possibility is that the agar plates contain too much agar and thus are too solid, which may cause the leaves to die. When the leaves become brown/ white within the first week or they look vitrified, the leaves could either be drying too much during the transformation procedure or, alternatively, they may have been submerged in the liquid medium during cocultivation.
Seed Transformation in Arabidopsis thaliana 15 7. Following a 24-h cocultivation, by which time the Agrobacterium have reached stationary growth, the mixture is filtered (Whatman #1) utilizing a Buchner funnel. The filters containing the seeds are placed in a fume hood for drying. It takes ca. 30 min to dry the seeds and within 1 h of drying they are sown. 8. For sowing, the seeds are scraped off the filter paper with a scalpel onto presoaked (modified Hoagland's solution) vermiculite in standard flats with drainage holes.
Cut off the apical parts of 8-12-day-old seedlings leaving 1-2 em hypocotyl attached. Transfer 10-20 seedling tips to a 90-mm petri dish with 25 ml RIM supplemented with an appropriate selective agent. 3. Score rooting and survival of the plantlets after 2-3 weeks of culture to determine the segregation ratio of the introduced selectable marker. 3 Comments After 3-5 days, transient expression of the gus-intron gene can be monitored using a histochemical assay (Jefferson 1987). An average of 3-6 GUS-expressing cells per explant (score 20-30 explants) indicates a successful transformation experiment.