Transformation of tobacco protoplasts with DNA entrapped in pH-sensitive liposomes

Abstract

A system for the transformation of tobacco mesophyll protoplasts using pH-sensitive liposomes was developed. Plasmid DNA (plGVneo23) encoding the NPT-II gene for kanamycin resistance was entrapped in pH-sensitive liposomes composed of dioleolphosphatidylethanolamine, cholesterol and oleic acid. These liposomes release their contents at low pH and are capable of delivering their contents into the cytoplasm of protoplasts. Kanamycin-resistant colonies were reproducibly recovered from transformed protoplasts at an average frequency of 1.62 x 10 -4 at pH 7.5. Plants regenerated from transformed cell lines were normal in appearance and were fertile. NPT-II activity was detected in leaf extracts of transformed, kanamycin-resistant plants and the presence of NPT-II DNA in the tobacco genome was shown by Southern blots. Analysis of self-pollinations and reciprocal crosses to non-transformed plants indicated that kanamycin resistance segregated as a dominant nuclear marker. Co-transformation of protoplasts with liposomes containing two selectable markers indicated that co-transformation occurred with a frequency of approximately 23%.

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

“…Xanthi transformed with the plasmid pLGVneo23 containing pnos-npt H (nos promoter sequence and the npt II structural gene) (Zhu et al 1990). Npt II encodes kanamycin resistance.…”

“…Preliminary tests indicated that the extraction buffer (Zhu et al 1990) interfered with the ELISA assay. Therefore, 0.25 M Tris buffer (pH 7.8) was used to extract protein from tissue for ELISA tests.…”

“…After several subcultures, calluses showed no detectable NPT II activity. Treatment of calluses with 5-azacytidine restored detectable levels of NPT II activity in calluses assayed (Zhu et al 1990).…”