A special kind of anniversary: 30 years of stability of the expression of an inserted gene (GUS) in cherry rootstocks shows high reliability for breeding

By a funny coincidence we came across an interesting discovery.

Our master's student MSC. PATRICIA BURILLO CARTAGENA needed for her transformation experiments with Olive (to create resistance to Xylella) a suitable plant tissue as a positive control to demonstrate successful transformation with a marker gene (uidA).

The GUS reporter system (GUS: β-glucuronidase) is a reporter gene system in plant molecular biology. The technique utilizes the uidA gene of Escherichia coli, which codes for the enzyme, β-glucuronidase; this enzyme, when incubated with specific colorless or non-fluorescent substrates, can convert them into stable colored or fluorescent products. The most common substrate for GUS histochemical staining is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). X-Gluc is hydrolyzed by GUS into the product 5,5'-dibromo-4,4'-dichloro-indigo (diX-indigo). The presence of the GUS-induced color DiX-indigo indicates if and where the gene has been actively expressed.

Our transgenic cherry lines, which we had regenerated at the beginning of our transformation work (at the time to create virus resistance in stone fruit) and have now maintained in vitro for 30 years, were ideal for this purpose.

In 1994 we succeeded for the first time in introducing marker genes (uidA and nptII) into cherries and demonstrating their function in the plant tissues. Starting from an embryogenic callus of Prunus subhirtella, we were able to regenerate entire plants via somatic embryos, in whose tissue the gene led to an expression-dependent blue coloration of varying intensity.

This material has allowed to study interesting aspects in relation to duration but also to stress response in expression levels of introduced genes and to publish several peer reviewed papers (See PBU website).

Fortunately, the transformation attempts with olives also led to a positive result, which contributed to a particularly successful conclusion to the Master thesis of our master student MSc. Patricia Burillo Cartagena.

About the publication:

da Câmara Machado, A., Puschmann M., Pühringer H., Kremen R., Katinger H. and Laimer da Câmara Machado M. 1995. Somatic embryogenesis of Prunus subhirtella autumno rosa and regeneration of transgenic plants after Agrobacterium-mediated transformation. Plant Cell Reports 14: 335-340.