Regeneration and Transformation Experiments in Apple
Successful introduction of foreign genes using a leaf disc procedure and cocultivation with Aqrobacterium tumefaciens requires a good regeneration protocol and tissue susceptible to infection. In our experiments, apple rootstocks M26 and M9 and the cultivar Mcintosh Wijcik were used. A good regeneration system was developed for M9 whereas a suitable protocol for the others had been previously developed. Transformation experiments were carried out with three wild type strains and three disarmed vectors harbouring ß-glucuronidase and neomycin phosphotransferase genes. Infection with three wild type strains (C58, A348, A281) did not cause gall formation. However, the binary vector pBH21 (35SGUS) x LBA 4404 resulted in transformed shoots of M26 and transformed callus of M9. This was confirmed histochemically using the substrate 5-bromo-4-chloro-3-indolyl glucoronide (X-Gluc) staining. Cefotaxime, used to control growth of the bacteria, had an effect simalar to cyto-kinin by stimulating shoot formation in vitro.
KeywordsWild Type Strain Shoot Regeneration Regeneration Medium Leaf Segment Transformation Experiment
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