The effect of selective agents and plant introns on transformation efficiency and expression of heterologous genes in pear Pyrus communis L.

摘要

The effect of selective agents on the efficiency of Agrobacterium-mediated transformation of pear was shown. The transformation frequency of the pear stock PS no. 217 by a binary vector carrying the nptll gene conferring kanamycin resistance was 0.4 or 3.1%, while the hpt gene for hygromucin resistance used as a selective marker increased transformation frequency to 6.2 (11.5%). In addition upon selection on hygromycin B, the proportion of pseudotransgenic regenerants considerably decreased. In four transformation experiments, twenty independent clones were recovered, and their transgenic nature was confirmed by PCR, histochemical, and fluorometric analyses of GUS activity. The presence of introns in the coding region of a heterologous gene was shown to influence the efficiency and stability of transgene expression in plant tissues. Fluorometric determination of GUS activity conducted for a period of two years demonstrated a threefold increase in transgene expression in the case that an intron-containing construct was used for transformation. The expression level was rather stable across several years the transformation procedure developed may be sued for successful expression of heterologous genes controlling agronomic characters in pear plants.