Stable transformation of embryogenic tissues of Pinus nigra Arn. using a biolistic method

The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l(-1). Resistant embryogenic tissue recovery required 10-12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.