Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum L.) with human lactoferrin gene and analysis of transgenic lines

Abstract

Aim. Obtaining of tobacco lines (Nicotiana tabacum L.) stably transformed with human lactoferrin gene (hLf), analysis of transgenic lines. Methods. Agrobacterium-mediated transformation of tobacco was carried out with the use of pBin35LF plasmid containing human lactoferrin gene under control of 35S promoter of cauliflower mosaic virus (CaMV 35S) and selective npt II gene encoding neomicyne phosphotransferase II providing the resistance to kanamycin. The selection of transgenic lines was carried out for 3 months on MS medium supplemented with 100 mg/l of kanamycin. Integration of lactoferrin gene was confirmed with the use of PCR with primers specific to hLf gene. The investigation of karyotype of transgenic lines was carried out after the staining of the root cells with 1 % solution of acetoorseine. Results. After selection, 2 transgenic tobacco lines with confirmed integration of hLf gene were obtained. The transformation efficiency was 6.4 %. The chromosome number in transgenic and control lines was 2n=48. Conclusions. The obtained transgenic tobacco lines have the similar morphology and karyotype to control lines, so they could be considered to use as plant systems for the expression of recombinant human lactoferrin.
 Keywords: human lactoferrin gene hLf, Nicotiana tabacum L., Agrobacterium-mediated transformation, PCR, chromosomes, transgenic plants.