Green fluorescent protein (GFP) supports the selection based on callus vigorous growth in the somatic hybrids Solanum tuberosum L. plus S. chacoense Bitt

摘要

Somatic hybridization based on protoplast fusion is a biotechnological tool enabling multiple resistance genes or traits to be transferred from wild relatives into crops. A key step for successful regeneration of hybrids is the selection of the heterokaryons. Selection can be done on specific culture media, by using intrinsic cellular or fluorescent markers, or specific traits of the fusion products, such as vigorous growth. More vigorous growth is one of the most common used criteria, since it does not require the use of mutants and allows faster selection of putative somatic hybrids. However, vigorous growth does not occur in all fusion combinations and often additional approaches have to be used (molecular markers, flow cytometry, chromosome counts, etc.) which are more costly and time consuming. There is, therefore, a need for the development of a faster, simpler, more cost-effective and reliable method for the selection of the heterospecific fusion products. Our study reports the effective use of the reporter gene gfp to confirm the vigorous growth as a reproducible selection for the somatic hybrids between Solanum tuberosum dihaploid line and the wild species S. chacoense. In vitro shoots of transgenic S. chacoense expressing the reporter gene gfp and a wild type dihaploid line of potato were used to isolate the mesophyll protoplasts. Electrofusion of mesophyll protoplasts and standard culture methods were applied. The S. chacoense gfp? protoplasts were unable to regenerate and when parental protoplast and putative somatic hybrid cultures were monitored, the GFP was expressed and visualized microscopically only in hybrid regenerants showing vigorous growth. The results confirmed that vigorous growth can be used to select the fusion products between these two species. This strategy might be applied to other fusion combinations to improve somatic hybrid selection.