Agrobacterium tumefaciens -mediated transformation of the apple rootstock M26 with GFP: increasing the efficiency by ultrasonication.

摘要

The objective of this study was to improve the efficiency of the genetic trans-formation of apple rootstock M26 by ultrasonication of leaf explants. Transformation frequency of Nicotiana tabaccum by Agrobacterium rhizogenes was increased by wounding caused by ultrasonication (Kumar et al., 2006), and we wondered if the transformation frequency of apple rootstock M26 could be increased by a similar treatment. Leaf explants of in vitro cultivated M26 plantlets were ultrasonicated for 0, 5, 15, or 60 s, respectively. Then the explants were co-cultivated with A. tumefaciens strain ATHV harbouring a binary vector carrying a gfp (green fluorescent protein) reporter gene. Explants were examined weekly under UV-light for GFP-fluorescence over a period of twelve weeks. GFP-fluorescence was monitored in the explants with respect to intensity and localisation. Furthermore, the vitality of the explants was recorded. The duration of the treatment with ultrasonication influenced quantity, intensity and localisation of GFP-fluorescence. The shorter the duration of the ultrasonication treatment, the more GFP-fluorescent spots were visible. On average, explants subjected to ultrasonication for 60 s showed fewer GFP-expressing spots. However, these areas were located in callus tissue more frequently and their fluorescence was brighter than the other treatment regimes. The higher rate of callus with GFP-fluorescence and regeneration of GFP-expressing shoots showed a positive influence of ultrasonication in comparison to samples without or with shorter ultrasonication treatments.